Team:St Andrews/Notebook
Notebook
June
Week 1:
Monday 7th June
First official day of our summer work! Everyone on the wetlab team worked on getting Covid-19 lateral flow tests for the lab work, and we did some work on finding funding grants.
Tuesday 8th June
Had a wetlab Microsoft teams meeting to discuss our upcoming lab work, AJ and Anna M. agreed to work on the iGEM Impact grant application. There was also a human practices meeting today, ideas were discussed on outreach, and potentail continuation of the CoralWare app developed by the 2020 team.
Wednesday 9th June
Maia, AJ & Julia
First official day with access to the lab and also the Hive (a study space within the biology department) today. We had a safety briefing from Dr. Magnus Alphey, and found that our lab supervisors would be Jacqueline (our PI), Dr. Simon Young, and PhD student Teresa Filipa Guerreiro MacHado.
We also had a Whole Team Meeting. We discussed stipends (Wellcome), the safety form (due June 25th), the Wiki, wetlab, and the Tuesday human practices meeting. Also made a list of everyone and their respective degree schools -- Chris will contact the departments and see if they can grant us any funding.
Thursday 10th June
Worked on organizing team finances, we emailed Jacqueline to ask her about the funding amounts from the Wellcome Trust. Also worked on preparing the presentation for the wetlab work plan.
Friday 11th June
Plan for another Human practices meeting next week. We worked on finishing the wetlab presentation and filling out biology department lab safety forms.
Saturday 12th June
Continued project and wetlab presentation work.
Sunday 13th June
Had a short wetlab meeting about how the wetlab presentation work was coming along. Discussed dates for the lab work to put on the Gantt chart, and other minor edits to the presentation slides.
Week 2:
Monday 14th June
After preparing the powerpoint last week, the wetlab team (+Shagun) presented the basics of our project to our three chosen laboratory supervisors, Dr. Jacqueline Nairn, Dr. Simon Young, PhD Student Teresa Filipa Guerreiro MacHado. We discussed the general aim of the project, full gene circuit, parts we need for laboratory work, and expected timelines (see the original presentation here: Initial Aims Wetlab Powerpoint .pdf). After our presentation, we talked with everyone about the structure of lab hours, safety, and initial plans for the project lab work.
Gantt Chart from our presentation to our lab supervisors.
Some of the initial lab work plans we discussed with our supervisors included:- UV assay ideas for testing prescence of shinorine
- Best to order parts (use IDT Gblocks, needed to get a cost code from the University)
- Locating past iGEM laboratory supplies
- Necessary laboratory supplies (pipettes, machines, supplies, etc.)
- Plasmid assembly
After the presentation, the in-person wetlab team met with Simon (who would be supervising us on Mondays and Tuesdays) to discuss parts and plasmid design. Through our researching through the 2020 St Andrews iGEM wiki page, we found out that the plasmids they had designed for the implementation of the Shinescreen gene circuit for the (Phase 1) project were not actual plasmids. They were theorhetical designs, optimized for the in-silico design of the project and not laboratory implementation. We realized that we needed to find and design our gene circuit to fit into actual plasmids. To discuss this, we then had a talk with Simon about:
- Plasmid assembly (found it was best to stick with one assembly method – so we save time)
- Gene expression (how the 1st inserted gene is normally expressed more)
- Cloning vs. expression plasmids
- What other University labs might be able to lend us for laboratory work
Tuesday 15th June Maia, Julia & AJ
We decided on using Gibson assembly for cloning. Therefore, time was dedicated to learning more about the workings of Gibson assembly.
We asked for everyone's matriculation numbers (for sorting out Wellcome funding), a key for the biology Hive building (study rooms), and a cost code from Lianne for ordering our parts. The UK/Ireland iGEM meetup was scheduled for Sunday, June 27th, and we planned to work on a short presentation for that (about our project).
We talked about ideas for how we would assemble the genes into our plasmids - watched part of the synthetic biology lecture (that Maia/AJ had) - it covered Gibson Assembly, 3A assembly, and cutting with restriction enzymes.
We all had a long discussion with Simon about the best plasmids, how to obtain the sugar intermediate (seduheptulose7-phosphate) for shinorine production, and E. coli codon bias. For plasmids, Simon told us that our final plasmid constructs would need to have similar restriction enzyme sites and be compatible with each other in the same cell (to avoid plasmid incompatibility).
We discussed best assembly methods for our plasmids too. The methods we considered were:
- Restriction enzymes
- Standard iGEM assembly (ISS)
- 3A assembly (antibiotics)
- Gibson assembly
Our first plan for assembling our genes was to use restriction enzymes to cut and paste our genes into our plasmids. Looking back through Maia and AJ’s second year synthetic biology notes we learned more about restriction enzymes. Specifically, we considered finding pairs of palindrome restriction enzymes, which are two enzymes that have the same overhanging sequences, thus leaving no gaps in basepairs when inserting our genes (ex: Spel and Xbal). However, the only issue with this method is that we would have needed to add in extra base pairs onto our genes to leave in enough room for a restriction enzyme site. And due to iGEM’s regulations of biobrick parts only containing the sequence, we figured that this method would not be the best. Another issue we encountered was that when assembling genes with restriction enzymes, scar sequences are left behind, and we wanted to avoid this problem. In the end, we talked with Simon about this and decided to use Gibson Assembly for making our plasmids (and inserting genes).
Reading more into the procedure, we learned that Gibson creates no scar sequences, only requires special primers and a mix, and can join together gene and plasmid sequences in one PCR reaction. We concluded that this was the ideal method for our team to use, as it looked to be the quickest and most efficient way to add in our genes (and still follow iGEM guidelines as well). For Gibson assembly though, we realized that we would need to design a new set of primers to use for the process. We had existing primers on Benching (provided via the 2020 team), and we aimed to work on making some new primers for Gibson in the coming days. Similarly, we also found that Benchling has a Gibson Assembly tool, so we experimented with that.
We got confirmation that we received the funds for a £1000 grant from the SeaChangers organization! They wanted us to make sure to keep them updated on our work and what we use the funds for.
Along with deciding on our gene assembly method, we discussed the best plasmid setup to use. We decided on finding plasmids with 2 individual cloning sites (a separate promoter, RBS, and terminator), so that we have the space for putting in our genes (and eventual thanogen parts). Another thing that we wanted in our plasmids was a protein tag – near where we would insert our genes so that we would be able to better see them when we start testing for gene expression.
We talked briefly about codon optimization and codon bias in the genes - but ours were okay (as that had been handled by the 2020 team). For Gibson Assembly we also discussed about how many reactions we might do, and that we will need a proofreading polymerase that can replicate large gene sequences. Simon also mentioned to us that we should do sequencing too after Gibson to check our gene sequences.
We decided to go with pETDuet-1 and pACYCDuet-1 for our 2 expression plasmids, and the DHQS/O-MT composite part, and ATPG and NRPS as separate genes (since they rate limit reactions). Though we agreed to keep in mind the size of our plasmids and make sure they each would be similar sizes (for ease of transformation). Simon went to ask around the bio department and see if anyone had these plasmids for us borrow.
Another important thing that we discussed was our intermediate sugar seduheptulose 7-phosphate (S7P), which is where the shinorine production pathway starts with our genes. We talked with Simon about how E. coli naturally produces this intermediate (via the pentose phosphate pathway) - it was available for purchase but cost way too much for our budget. In light of this, we started to brainstorm some ideas about how to get enough S7P without having to buy it. Some ideas we had:
- Supplement E. coli with a sugar (a few steps before S7P) and they produce it naturally
- Order in shinorine from a chemical company
- Increase S7P production in E. coli (gene editing)
However, the issue with this idea is that S7P is an intermediate compound, and it has a high turnover rate. This meant that E. coli would use up S7P rather quickly, and there would be competition for S7P between the E. coli pathway and our inserted shinorine pathway. To combat this, we considered the idea of potentially changing the gene regulation or knocking out specific genes in the E. coli. But this was not a plausible action for us in the lab, and may have impacted the viability of the organism. Eventually we can to the conclusion that more research needed to be done on this.
Wednesday 16th June
Maia, Julia & AJ
We decided to order our gene in the form of gblocks from IDT, we then checked our gene sequences using IDT’s tool. All of our genes were satisfactory for manufacture, apart from NRPS which had a section of bases within it that increased the difficulty score above 7 which would have made out order hard to make and take a long time to. This was due to a stretch of sequence having too many guanine bases next to each other starting at base 1111.
To avoid this, we changed the sequence from GGGGGGGTGGGCG to TTA GGC GGT TGG GCC which was also codon optimized for the abundance of nucleotides in E. coli. This change was then re-checked with IDT tool, and the difficulty score had been reduced enough that we were fine to use this in our order.
All the genes were then checked to have the ATG start codon which is needed in E. Coli.
Jacqueline came in today (she supervised on Wednesdays and Thursdays), and we talked with her about our plasmid choices and looking over our Gibson setups. She mentioned that if we were able to get plasmids from researchers in the biology department we would need to set up a plasmid stock (would need to know storage temps and concentration). We sent out emails today to ask if any biology researchers had either bacteria or the pETDuet-1 or pACYCDuet-1 plasmids (Prof. Terry Smith, Prof. Malcom White, Dr. Tracy Gloster, Dr. Clarissa Melo Czekster, and Dr. Rafael de Silva). Found out also that Margaret Wilson is the main organizer for payment of the Wellcome stipend money.
We also considered other ideas of how to get Sedohetuplose7-phosphate. The main ideas were that we maybe would try to find methods to mimic S7P (substrate mimics). We made a new document about this, and used the Brenda enzyme database a bit (to check each of our shinorine genes).
We also talked a bit about some E. coli strains that would be okay to use for gene expression (BL21, Rosetta 2, etc.). Heard back today also that Rafael and Malcolm have things for us (BL21, etc.). We worked some more on the iGEM safety form.
We did some more work on analyzing our gene parts for the shinogen and checking if there were any issues. After looking at the DHQS and O-MT composite part again, we asked Lawrence (from 2020 St Andrews team) if there was an internal ribosome entry site (IRES or RBS) coded onto the DHQS O-MT composite part. Other than that inquiry, there were no major issues with that part except a small hairpin potential. We found (using the IDT Gblocks order site) that the NRPS gene had 13 bases that had too high of a guanine (G) content (starting at bp 1111 of the gene). We worked to fix this issue using the DNA BLAST program to check that the bases we change still code for the same amino acids. The original amino acids (that we wanted to keep) were Leucine, Glycine, Glycine, Tryptophan, and Alanine (the base pairs were TTG GGG GGG TGG GCG), and had a GC content of 84.6%. We resolved this issue, and the DNA sequence changed to TTA GGC GGT TGG GCC. The ATPG gene was okay, and we didn’t need to change its bases for a different E. coli start codon. After finishing labeling the DHQS and O-MT composite part, we found that it did not have any issues with start codons or GC content. We used BLAST suite to compare nucleotide sequences, and this helped us with labelling parts.
Thursday 17th June
Maia, Julia & AJ
Primer design for Gibson assembly began. Tried to do this using Benchling’s Gibson assembly Wizard tool, however, couldn’t get the genes aligned to the correct cloning sites in the plasmids. It was also decided that ATPG and NRPS would be inserted into pACYCDuet-1 separately at two different cloning sights and the DHQS_O-MT composite part would be inserted into pETDeut-1.
We worked more on Gibson primer design today and made a new doc on OneDrive for it. The pETDuet-1 plasmid had 5,420 base pairs, and pACYCDuet-1 had 4,008 bases. After adding in the shinogen genes, the plasmids were about 8,000 bp (pACYCDuet-1) and 7,500 bp (pETDuet-1). We also wrote a follow up email to Malcolm asking what specific plasmids and bacterial strains he had.
Image of the initial plasmid design using Benchling Gibson Assembly Program (ATPG and NRPS on pACYC).
We also worked some more on the safety form (due June 25th), and asked Dr. Magnus Alphey (head of security for biology) if he would give us a talk on fire safety (refresher and for Anna and Yoricka, who had been quarantining an not in the lab yet). After being awarded the Promega grant, we also continued to work on making up a list of supplies we needed for the lab and sent an email to Simon to check over our Gibson assembly primers. We also worked some more on our UK/Ireland iGEM meetup presentation.
We found that we wanted the genes for pACYCDuet-1 to be separately expressed, so we needed to redesign some of our Gibson primers (as we originally had the genes next to each other). We also needed to change the location off the DHQS and O-MT composite part so that it fits in the reading frame (as it has its own promoter, RBS and terminator already).
Friday 18th June
Maia (& Terresa – supervisor)
Primer design was corrected using the NEBuilder tool online, we were able to insert each gene into the correct location by making the start position of each plasmid the base after the end of the HIS or S tag (which were within the cloning site) and the end position the last base of the appropriate tag. This then meant that our genes were inserted into the cloning sites and would be eiter HIS or S tagged for future analysis.
Maia, Julia & AJ
The decision was also made to insert the DHQS and O-MT genes into two different cloning sites due to the lack of terminator after the first gene in the composite part, this was just to allow us to establish initial expression and may be changed further down the line.
Today we had lots of help from Teresa with Gibson Assembly design (she supervised us on Fridays). She suggested that we use the NEBuilder website for designing our primers instead of Benchling. She had used that program before, and done many Gibson assemblies for her PhD, and was a great help to us!
We sent our finished powerpoint to Jacqueline for review before the UK/Ireland iGEM meetup. Stipends were also discussed a bit more today, and we has Dr. Chris Hooley (our other PI) to ask the Biology, Chemistry, Computer Science, and Psychology departments if they would be able to provide us funding. Teresa also told us that she would be able to get us BL21!
After our initial Gibson Assembly work, we found that the DHQS and O-MT composite part we planned to use didn’t work in the setup. Since the part had its own promoter, and where we wanted to put it in the plasmid had a T7 promoter, we weren’t able to use it. We asked Lawrence if it was okay to use the T7 promoters on the plasmid for the shinogen genes for expression, and he said that would be okay. So we decided to just use the genes themselves in our assembly. Though by not using the composite part, the genes may not be the most optimal in expression, Lawrence told us that we should still have some. That was okay with us! After that change we figured out that we would need to do two separate Gibson assemblies to insert all the shinogen genes (as no genes were right next to each other). This meant that we would need to order 16 total primers (4 for each Gibson reaction). We sent these primer and gene setups to Simon and Jacqueline to look over.
In summary, our planned order for gene parts for the shinogen system included the 4 Gblocks and 16 primers. We also figured out that we would need:
- a Gibson Assembly kit (on NEB)
- a plasmid miniprep kit (on Promega)
- a high fidelity polymerase (we found Pfu on Promega)
- LB Broth (in the biology department store)
- DPN1 (for cleaning products, on Promega)
- And a PCR cleanup kit (on Promega)
- Antibiotics (ampicillin and chloramphenicol, for testing expression)
Continued to work on various aspects of the project.
Sunday 20th June
More work on the project.
Week 3: Monday 21st June
Maia, Julia & AJ
Initial drafts of the safety form (due at the end of this week) were continued and read over. We also started to finalize our order for Promega (we received a $2500 grant in vouchers from them) and planning the reagents, kits, etc we would need in the future. We also looked into a Gibson assembly protocol that would be appropriate for our genes and decided on the protocol (can be found in the protocols section of OneDrive). We heard back from Shirley (who works within Malcolm White’s lab) that they are willing to lend us expression E. coli strains and our plasmids (pETDuet-1 and pACYCDuet-1).
We also had a long talk with Simon about our protocol outline for work in the lab. We talked about:
- Obtaining glycerol stocks of our plasmids
- Process for plasmid minipreps
- Gibson Assembly and transformations
- General guidelines for protein expression and purification
Work continued on the safety form - we took some pictures of our laboratory space and worked on writing up our responses to the form questions.
The pictures of our lab for the safety form! We were very grateful to have access to a large teaching laboratory.
Wednesday 23rd June
Maia, Julia & AJ
Completed safety form and checked it was saved correctly. The genes and primers were ordered from IDT. We began to construct plasmid maps which corresponded to the restriction digests we hoped to carry out to confirm the insertion of our genes and the fragments these digests would produce.
PACYCDuet-1 plasmid restriction enzyme map.
Our plates containing our plasmids were also streaked by Dr Shirley Graham, who we have obtained the plasmids from.
Thursday 24th June
Maia, Julia, AJ & Anna M (for recording)
Reordered our presentation for the UK meetup this weekend, held by KCL. After this we continued to work on the Promega order form. We started to plan all of the protocols we would need to conduct over the entire project.
We also changed one of the plasmids from the pACYCDuet-1 plasmid to the pRSFDuet-1 plasmid as the latter had the Kanamycin resistance gene which is easier for our team to get a hold of and is more abundant in the University stores. These plasmids are identical apart from the resistance gene and so there was no need to change primers.
The plasmids and restriction enzyme maps were re-done to incorporate the change in plasmid, luckily everything matched up perfectly.
Friday 25th June
Maia, Julia & AJ
We continued planning protocols for next week and managed to get a starter protocol for each procedure we need to conduct, these will probably be edited and optimised for our work as we go.
The Promega order was placed! Teresa also picked up our plate streaked with DH5a (containing pRSFDuet-1) from Shirley, and our NEB Gibson Assembly kit came in the mail today as well.
Saturday 26th June
Worked on our project and prepared for the UK/Ireland iGEM Meetup.
Sunday 27th June
The UK/Ireland iGEM Meetup was held today on Zoom. Had a nice talked delivered from Jenny from Promega about job opportunities for everyone who was doing iGEM. There was also a short Q&A session too. Then the rest of the meeting was for project presentations and chatting between all the attending teams. We heard from the other teams, and our project presentation was also shown! The five other attending teams were:
King’s College London (KCL)
Thanks again also to KCL for hosting – it was great to meet everyone on some of the iGEM teams this year!
Week 4:
Monday 28th June Maia, Julia, AJ, Anna S & Yoricka Finalised the protocols we will need and made sure we had enough information to conduct each procedure. Rewatched the gene circuit presentation from Lawrence/Caius (2020 team) for a better understanding of the gene circuits. We also attended the iGEM automation workshop which was super interesting and showed us robotic solutions we had never thought of before. Tuesday 29th June Maia, Anna, AJ, Julia & Yoricka Today we went over to the lab and organised it into station e.g., for making up solutions, running gels and culture work. Also had a talk with Simon about some more ideas about optimising production of seduheptulose 7-phosphate (S7P). Some of the findings we discussed: S7P doesn’t seem to be essential in the pentose E. coli pathway, so hopefully as long as we keep the E. coli happy they should be able to express our genes May be able to stimulate the pathway using hydrogen peroxide? In mammillian cells apparently their pentose pathway is stimulated by UV light This could be something to consider in the future if our expression trails don’t work Potential gene addition here? We also need to make sure to use as many different E. coli strains as we can to optimise protein expression Overall this was a hopeful talk, but quite a while in the future so we agreed to keep it in mind as we focused more on planning for the lab. Maia I also looked into Thanogen kill switch parts and organised which plasmids each gene needs to go into. Yoricka I looked more into the issue of testing the presence of Shinorine. The thing is that even if we see that our product is UV-protecting, that won’t necessarily tell us that there is Shinorine, as other genes also offer UV-protection (like Mysosporine glycine which is the actual UV-protecting part and gets converted into Shinorine by NRPS enzyme). So, I started to look for compounds with similar wavelengths absorption as Shinorine, which could be bought and use to test Shinorine presence. Buying Shinorine itself is not a great option as it is quite expensive. Wednesday 30th June
Maia
I started the construct of the Gibson assembly for the Thanogen parts and designed the primers for the light sensing component.
Maia, Julia, AJ, Anna, Yoricka
We all conducted research into previous work using Thanogen systems, we had our weekly team meeting in the evening too. Also went over to the Medicing Building and picked up lab coats for us to use.
Yoricka
I found that some of the colourless carotenoids, that are also in cyanobacteria, can have very similar wavelength absorption as Shinorine. After comparing the prices, Lutein seems to be rather available and cheap and some of its isomerases have practically the same wavelength absorption as Shinorine. They should also be completely harmless to all the compounds as well as to humans and the sea. I put some graphs and tables into the Complete protocol document.
July
Thursday 1st JulyMaia
Finalised all of the Gibson assemblies for the Thanogen parts and the primer design for inserting them into the plasmids which should have the Shinogen parts are already in.
Maia, Julia, AJ, Anna, Yoricka
We all conducted research into previous work using Thanogen systems.
Julia
I did some brainstorming on an idea for ‘excellence in another area’ for iGEM Gold Medal Criteria #7. It was a general manufacturing plan for shinorine (also made a new document with my ideas). Is another potential 4th gold criteria we may go for in the future!
Yoricka
I studied the PPP in more detail to see what will happen in case of S7P shortage and how that could be overcome or prevented. PPP is one of the many pathways that glucose 6-phosphate (G6P) can enter and according to the research on it, it seems that G6P is highly responsive to the environment in that sense and can easily opt out between the pathways it will enter. This suggests that in the higher need for S7P, G6P could possibly opt out for the PPP more, however, there isn’t any paper that would explicitely claim this, as researchers have mostly be concerned with there being enough G6P for the Embden-Meyerhof Pathway (which is one of the two possible pathways of G6P in bacteria), which is basically the other side of the issue.
Friday 2nd July
Maia, Julia, AJ, Yoricka & Anna
We all worked on applying to grants and funding. Our order from Promega also came in today!
Yoricka
After more S7P research, I am positive that the only thing we should be concerned about is just lack of glucose. If there is enough glucose, it can then enter the PPP very easily and in either the normal way through dehydrogenation, or through a “gluconate shunt”, which allows it to skip over couple of steps and makes the PPP faster. The only issues are all to do with glucose starvation, which prevents the gluconate shunt from happening, and it activates certain proteins that further adjust the direction of glucose, making it less likely to enter the PPP. In conclusion, if we have enough glucose, it should all go well as the system will produce enough S7P, even if we are using a lot of it. We just need to make sure we have enough glucose, which Simon said might be achieved just by making sure our bacteria is happy and well fed, but there are possibilities to look for G6P, which looks much cheaper than S7P.
Saturday 3rd July
More work on the project and preparing for in person wetlab work next week!
Sunday 4th July
Continued project work.
Week 5:
Monday 5th July
Maia, Julia, AJ, Yoricka & Anna
Setting up the bacteria to grow overnight culture
Materials:
Agar plates one with E.coli bacteria with pET Duet-1 plasmid, and one with E.coli bacteria with pRSF Duet-1 plasmid
Antibiotics (ampicillin and kanamycin)
LB broth
Conical tube
Tuesday 6th July Maia, Julia, AJ, Yoricka & Anna Plasmid Miniprep To isolate the plasmid from E.coli (it removes proteins, RNA, and other contaminants and purifies the plasmid DNA). We did this for both the successful pETDuet-1 and pRSFDuet-1 overnight cultures.
Miniprep Kit:
Cell lysis Buffer
Neutralization Solution
Endotoxin Removal Wash
Column Wash
Elution Buffer
Minicolumns
Collection tubes
Preparation: Dilute Column Wash Solution with 95% ethanol => For 10 (minimum) preps (4ml 95% ethanol) Process:
600ul of bacteria in the LB broth to a 1.5ml eppendorf tube
Add 100ul of Cell Lysis Buffer and mix by inverting it 6 times -> turns blue
Add 350ul of Neutralisation solution (from fridge (4-8C) and mix by thorough inverting -> turns yellow
Centrifuge, max speed, 3min
Transfer it (~900ul) to minicolumn with a pipette
Put minicolumn to a collection tube and centrifuge (max speed, 15s)
Discard the flowthrough and put it back to the same collection tube
Add 200ul of Endotoxin Removal to the minicolumn -> centrifuge (max speed, 15s)
Add 400ul of Column Wash -> centrifuge (max speed, 30s)
Transfer the minicolumn to a new, clean 1.5ml microcentrifuge tube -> add 30ul of Elution Buffer directly and let it stand for 1min in room temperature
centrifuge at maximum speed for 15 seconds to elute the plasmid DNA -> cap the microcentrifuge tube and store it at –20C
We set up overnight cultures with DH5a containing pETDuet-1 and pRSFDuet-1 to make up a glycerol stock of the cells. We had 3 tubes total, 1 for a control (media only), 1 with ampicillin and pETDuet-1, and 1 tube with kanamycin and pRSFDuet-1.
For each tube we added 5 ml of LB Broth in a 15 ml conical tube, and for each of the tubes with DH5a we added 5ul of the corresponding antibiotic, and 1 colony of bacteria. We put these on the shaking incubator overnight.
Methods:
We diluted the antibiotics to create their ideal concentrations, which is 100ug/1mL for ampicillin and 50ug/1mL for kanamycin. We diluted the antibiotics to get 5mL of each stock.
We took a conical tube and transferred 10mL of the LB broth into it using a pipette.
We added 10uL of each antibiotic into the conical tube.
Using a sterile, plastic pick, we selected a single bacterial colony with the pET Duet-1 plasmid from the agar plate and transferred it into the conical tube with the LB broth and antibiotics and swirled it to mix.
We repeated the process for the bacterial colony with the pRSF Duet-1 plasmid.
To set up a control, we have again put 10ml of LB broth and 10uL of each antibiotics stock into a new conical tube but without any bacteria.
All three tubes were closed with their screw lids and placed into a shaking incubator at 37°C overnight (16 hours).
Wednesday 7th July
Maia, Julia, AJ, Yoricka & Anna
Glycerol stock for storing the plasmids (we made couple of tubes just in case, I’m not sure how many exactly now)
Add 500ul of the overnight culture to 500ul of 50% glycerol (100% glycerol diluted in dH2O) in a 2ml screw top tube and gently mix
We only had 40% glycerol, so our amounts were 620uL of glycerol and 380uL of the culture
Freeze at –80C
We also used the nanodrop machine to quantify our plasmid DNA samples from the previous day
Results:
Thursday 8th July
Maia, Julia, AJ, Yoricka & Anna
The primers have arrived, so we rehydrated and diluted them
All the tubes were briefly centrifuged before opening
Resuspended in EB buffer to a 100µM stock concentration
Stored in Eppendorf tubes in the freezer at –20oC
Preparing agarose gels for the PCR
Agarose and TAE buffer put into a conical flask
Microwaved for 30 seconds, swirled around, put back in the microwave and repeated until the agarose visibly dissolved
Flask then set aside to cool until is cool enough to touch (but still warm) and then poured into the casting unit and let sit to thicken and form the gel
Friday 9th July
Maia, Julia, AJ, Yoricka & Anna
Not in the lab that day, just in the Hive planning PCR conditions for the following week.
Saturday 10th July
Continued work on the project.
Sunday 11th July
More work on the project!
Week 6: Monday 12th July
In the laboratory:
12/07/21 - Anna, Maia, Julia, AJ and Yorika
PCR was conducted of plasmid pET-Duet-1. The PCR mix contained DNA (3.6 µl, 687 ng/µl), primer pET-Duet-1_fwd (1 µl, 10 µM), primer pET-Duet-1_rev (1 µl, 10 µM), GoTaq Long PCR Master Mix 2X (25 µl) and nuclease free water to a final volume of 50 µl. PCR of the plasmid was conducted twice for consistency. The concentrations, temperatures and timings of each step are detailed below.
PCR was conducted of plasmid pRSF-Duet-1. The PCR mix contained DNA (4.1 µl, 1204 ng/µl), primer pRSF-Duet-1_fwd (1 µl, 10 µM), primer pRSF-Duet-1_rev (1 µl, 10 µM), GoTaq Long PCR Master Mix 2X (25 µl) and nuclease free water to a final volume of 50 µl. PCR of the plasmid was conducted on 3 samples at a temperature gradient*. The concentrations, temperatures and timings of each step are detailed below.
Our swag kit from iGEM HQ also came in the mail today! We posted about it on our social media accounts and took pictures with it in the lab. 
Tuesday 13th July
13/07/21 - Anna, Maia, Julia, AJ
Gel electrophoresis was conducted for the 5 plasmid PCR samples from yesterday, pET-Duet-1 and pRSF-Duet-1 pre-PCR and on 2 ladders (100-1000 bp and 100-10,002 bp) using a 6X DNA loading dye with bromophenol blue and xylene cyanol FF. The gel was ran in a 1X TAE buffer (40 mM Tris (pH 7-8.5), 2 mM sodium acetate, 1 mM EDTA) on a 1% agarose gel prepared on 08/06/21, for 45 minutes at 80 V.
The gel was incubated at room temperature, protected from light, whilst shaking, in 1X Diamond nucleic acid dye in 1X TAE buffer for 15 minutes, and then for a further 15 minutes when visualisation was not possible after this time. The gel was imaged using high intensity UV light (λ = 302 nm).
The gel after 15 minutes (visualisation is not good). And we imaged the gel on a plastic tray (which we used to dye the gel), and this may have impacted the imaging quality. From here on out we decided to image the gel directly on the glass screen (no interference from plastic).
The gel after 30 minutes. Imaging went a lot better!
The pre-PCR plasmids indicated the presence of DNA fragments (which corresponds to the previous Nanodrop results with the slightly higher than optimal 260nm:280nm ratios), but this is not an issue going forward. The band of each plasmid PCR products correspond to their respective base pair numbers (pET-Duet-1 at 5420 bp, pRSF-Duet-1 at 3829 bp). The optimal annealing temperature for plasmid pRSF-Duet-1 was found to be 65 °C. PCR of pET-Duet-1 showed a higher intensity signal, indicating more product was formed than for pRSF-Duet-1 so PCR will be repeated for pRSF-Duet-1, using a higher concentration of plasmid DNA in the mix.
Wednesday 14th July
In the laboratory:
14/07/21 - Anna, Maia, Julia, AJ and Yoricka
PCR was conducted of plasmid pRSF-Duet-1 using a higher concentration of DNA than previous PCR of this plasmid. The PCR mix contained 5 ng (8.2 µl, 1204 ng/µl) of DNA, primer pRSF-Duet-1_fwd (1 µl, 0.2 µM), primer pRSF-Duet-1_rev (1 µl, 0.2 µM), GoTaq Long PCR Master Mix 2X (25 µl) and nuclease free water to a final volume of 50 µl.
The PCR products from Monday were cleaned up by combining in equal volume with membrane binding solution (4.5 M guanidine isothiocyanate, 0.5 M potassium acetate (pH 5.0)) before transferring to a minicolumn. After 1 minute incubation, they were centrifuged at maximum speed for 1 minute and flowthrough was discarded. The bound DNA was washed with membrane wash solution (10 mM potassium acetate (pH 5.0) 80% ethanol 16.7 µM EDTA (pH 8.0)) (1x 700 µL, 1x 500 µL), centrifuged for 5 minutes and then for a further 1 minute after discarding flowthrough. Nuclease free water (50 µL) was added to the minicolumn which was incubated for 1 minute and subsequently centrifuged for 1 minute. The eluted DNA was collected and stored at -4°C.
Our genes arrived from IDT. The dried genes were briefly centrifuged before combining with elution buffer (100 µL), vortexing and incubating at 50 °C for 20 minutes. Each of the 4 gene solutions were aliquoted into 4x 10ng / µL stocks and stored at -20°C.
Planning was discussed for the PCR of each gene, mainly regarding sourcing of a viable polymerase but also annealing temperatures and timings.
Thursday 15th July
In the laboratory:
15/07/21 - Anna, Maia, Julia, AJ and Yoricka
Dr Jacqueline Nairn managed to supply Pfu DNA Polymerase, as well as Pfu DNA Polymerase 10X Buffer to us from stocks in the Biology department. She also supplied a printed-out sheet of the Complete Pfu DNA Polymerase Protocol.
We rehydrated the genes, following both IDT manufacturing instructions and advice from our supervisors. We rehydrated each gene (DHQS, O-MT, ATPG, and NRPS) to have a working stock of 10 ng/µl.
Our genes arrived from IDT today! In preparation for the PCR of ATPG (1378 bp), the working stock of each primer (4 µL, 10µM) was diluted by a factor of 5 using nuclease free water (16 µL). PCR of ATPG (1378 bp) was then conducted. The PCR mix contained the now-diluted upstream and downstream primers (5 µL each, 2 µM), dNTP mix (1 µL, 10mM each), nuclease-free water (23.5 µL), ATPG (10 µL, 0.1 µg / 50 µl PCR), Pfu DNA Polymerase 10X Buffer with MgSO4 (5 µL), Pfu DNA Polymerase (0.5 µL, 1.25 units / 50 µl PCR), giving a final volume of 50 µL. The programmed temperatures and durations of each step are detailed below.
PCR of gene NRPS was conducted overnight. The PCR mix contained the now-diluted upstream and downstream primers (5 µL each, 2 µM), dNTP mix (1 µL, 10mM each), nuclease-free water (23.5 µL), NRPS (10 µL, 0.1 µg / 50 µl PCR), Pfu DNA Polymerase 10X Buffer with MgSO4 (5 µL), Pfu DNA Polymerase (0.5 µL, 1.25 units / 50 µl PCR), giving a final volume of 50 µL.
We also ran a gel for pRSF PCR products from 14/7/2021 (2 samples with a 65 C annealing temperature). We used a 1% gel (made on TDB), and the concentrations of the plasmids, dye and ladders added are as follows:
100 bp ladder (5 ul ladder, 1 ul loading dye)
PRSF plasmids (1 ul PCR product, 1 ul loading dye, 4 ul deionized water)
10 kb-100 bp ladder (5 ul ladder)
With samples that used loading dye we made sure that the dye was in a 1/6 dilution, as the loading dye originally came 6x concentrated.
GEL MAP
1 – 100 bp ladder 2 – none 3 – pRSF PCR #1 (from 14/7/2021) 4 – none 5 – pRSF PCR #2 (from 14/7/2021) 6 – none 7 – 1kb (big) ladder Sponsorships:
Today, Dr Christopher Moffat from Promega (UK) visited our wetlab team at St Andrews. This was very exciting, since Dr Moffat was actually supervised by our Wetlab supervisor (Dr Nairn) during his PhD. He was very generous, offering the opportunity to ‘shadow’ him at Promega, and the team had a long discussion with him about the possible careers a Bioscience graduate can have outside of research. He also mentioned that the Promega website has a Student Resources Centre, which could help us if we ever need extra guidance on molecular biology techniques during our project/ university career.
Friday 16th July In the laboratory: 16/07/21 - Anna, Maia, Julia, AJ and Yoricka Dr Frances der Weduwen (an associate lecturer in Cell Biology and Biochemistry at the University of St Andrews) came to supervise us today, since Teresa MacHado has moved to London for the rest of the summer.
PCR of O-MT was then conducted. The PCR mix contained the now-diluted upstream and downstream primers (5 µL each, 2 µM), dNTP mix (1 µL, 10mM each), nuclease-free water (23.5 µL), O-MT (10 µL, 0.1 µg / 50 µl PCR), Pfu DNA Polymerase 10X Buffer with MgSO4 (5 µL), Pfu DNA Polymerase (0.5 µL, 1.25 units / 50 µl PCR), giving a final volume of 50 µL.
PCR machine malfunctioned with a heating error so may have to repeat after checking gels.
Saturday 17th July Sunday 18th July Today, Shagun, Chloe, Anna M, Weston, and AJ held a meeting to discuss progress towards the promotional video and the Integrated Human Practices, Partnership, and Education and Communication gold medals. Key points: Promotional Video: 90 seconds of the video are complete – these can be found on our shared OneDrive. Shagun simply needs to add another 3 seconds of animation, and attach credits as well as the 3 second intro video provided by iGEM at the beginning. The video should be complete by Wednesday – so if the rest of the team could have a look at the progress made so far and give feedback, that would be extremely useful. After the final checks on the video during our Wednesday weekly meeting, Shagun will make final changes if necessary, and then send the video off before the Friday (23rd July) deadline. Education and Communication: Chloe has received confirmation that she can put file the ethics applications through the Psychology department of the University of St. Andrews. She will complete the forms in the next few days. Regarding Education, we hope to target three demographics: Primary School children, Vegans, and Mum and Toddler groups. The consent for conducting workshops with Primary School children in St Andrews should go through once Chloe has completed the forms. With regards to the primary school children demographic, we hope to acquire laboratory materials from the Marine Biology department of the University, supply these to target schools within St Andrews, and deliver workshops/ practical classes to the children (early next semester) using these materials. This plan assumes that we will be able to deliver classes in person- if not, we should have some lectures/virtual classes prepared as a back-up. With regards to Vegans, we hope to collaborate with the Vegan Society of the University of St Andrews. We believe we do not need to fill in an ethical consent form in order to collaborate and host an event with the Vegan Society. Shagun will email the Vegan Society about a potential collaboration (taking place at the beginning of next semester) tomorrow. If Vegan society agree to collaborate, then AJ, Shagun, and Yoricka should create a PowerPoint/workshop to present to the society (the event will be open to all students in the university) regarding sunscreens, and what actions people can take to reduce their environmental impact in terms of sunscreen use. With regards to Mum and Toddler groups, Chloe will complete the ethical approval forms in the upcoming days. With this demographic, we hope to deliver a workshop to mothers, explaining 1) why applying sunscreen to your/your baby’s skin is extremely important, 2) how some sunscreens have very detrimental environmental effects, 3) how sunscreens can have harmful consequences to the human body (?), 4) the sunscreen alternatives that this demographic can use (we will both promote our own product, as well as other products that are already commercially available). Integrated Human Practices: So far, we have conducted two interviews: one with a bioengineer from YUN (a company specialising in probiotherapy - specifically acne treatment), and one with the CEO of mama KULEANA (a company that sells reef-safe sunscreen). We did not previously complete ethics approval forms for these, although now we believe that this is something we should have done – we'll fill these out and send them over to the Psychology department. We have more interviews lined up. We’ll need to apply everything we have learnt/will learn in the interview to further our project, both with respect to wetlab work, as well as human practices, advertising, and outreach. Partnership: We’re aiming to achieve a gold medal in partnership with KCL. KCL are creating educational material that they’ll deliver to secondary schools in South London, and they’re also hosting a synthetic biology competition amongst these schools. We will help them by producing pre-recorded lectures about synthetic biology, and increasing the outreach of their synthetic biology competition to include Scottish secondary schools. Hopefully, KCL will be able to produce educational material for us too, regarding the practical workshops that we’ll be delivering to primary school children throughout St Andrews. We’re aiming to achieve a silver medal by collaborating with Manchester. Julia will feature on an episode of Manchester’s podcast, talking about the application of synthetic biology to cosmetics.
Wiki:
Weston is asking the team to send individual headshots, so he can upload photos of us onto the website.
He needs the rest of the team to send informative content which he can then upload + format onto the website.
Action points:
Anna to keep working on the Entrepeneur Project.
Shagun to start creating the poster when promotional video is complete.
Weston to keep working on the wiki and to add content when supplied by the rest of the team.
Chloe/Shagun to finalise ethics for outreach/human practises projects.
Chloe to contact KCL concerning partnership ideas.
Chloe to figure out what content is required on the wiki to achieve medal criteria.
Shagun to email Vegan Society, asking whether we can collaborate with them by giving an informative workshop/lecture on sunscreens.
Yoricka and AJ to start making informative content if / when Vegan Soc agree to a collaboration.
Everyone to research viable, environmentally-friendly sunscreen alternatives that our iGEM team can promote to Mum and Toddler groups, Vegans, and School children.
Everyone to start writing down content to be uploaded onto the wiki (especially content relevant to medal criteria).
Human Practices + Education team to start filling in the notebook/continue filling in their daily contributions in the excel spreadsheet.
Everyone to look over the promotional video on OneDrive, and inform Shagun of any suggestions they have.
Week 7:
Monday 19th July
In the laboratory:
DHQS PCR
Making up 2% Agarose Gel for Electrophoresis
PCR cleanup ??
Electrophoresis
Visualising gene fragments under UV light
O-MT band didn’t show up under UV light, most likely since the PCR (carried out on Friday) failed to complete (there was an error code when we came to collect our PCR product, although it was shown to work at lunch). Promotional Video:
Today the wetlab team viewed the (almost complete) promotional video found on the OneDrive. The team highly enjoyed the video and were very impressed by Shagun’s animation efforts. AJ collected the following three suggestions from the team:
Add some background music to the video
Include photos of each team member when we introduce our team in the video
When discussing coral bleaching, animate the corals to turn white
When replying, Shagun stated that she’d be happy to incorporate photos of each team member, and that she would add background music if we managed to find some suitable royalty-free music. She also said that she had tried to animate the corals to turn white previously, but this proved to be very difficult. She will try again when the whole video is complete, however we may have to end up leaving the animation as it currently stands in the frame.
During the wetlab team’s lunch break, AJ and Julia managed to find some suitable royalty-free music that could be included in the promotional video. Julia sent a link of this music to Shagun.
Wiki:
In the evening, AJ wrote a rough draft for the Project Description (to be uploaded onto the wiki). She also highlighted the Project Description Criteria at the bottom of the document, and asked the rest of the team to proof-read her work and add any suggestions.
Tuesday 20th July
In the laboratory:
20/07/21 - Anna, Maia, Julia, AJ and Yoricka
PCR of DHQS:
PCR of DHQS was repeated since the dyed agarose gel (run Monday 19th July) showed too many bands for DHQS under UV light following electrophoresis. To improve upon this previous result, three PCR mixes were created this time: these were placed at different points along a temperature gradient (centred at 63 °C, with a 12 °C range) during the annealing stage of each PCR cycle. In each PCR mix, the volume of DHQS was reduced from 10 µL to 1 µL (thus the volume of water was adjusted from 23.5 µL to 32.5 µL). The denaturation step (in each of the 35 cycles) for each mix was reduced from 1 minute to 30 seconds.
Each PCR mix contained the diluted upstream and downstream primers (5 µL each, 2 µM), dNTP mix (1 µL, 10mM each), nuclease-free water (32.5 µL), DHQS (1 µL, 10 ng), Pfu DNA Polymerase 10X Buffer with MgSO4 (5 µL), Pfu DNA Polymerase (0.5 µL), giving a final volume of 50 µL.
Quantifying the DNA using the NanoDrop spectrophotometer. (put in graph image here!)
Results:
ATPG: 104.1 ng/µL
260/280 ratio: 1.93 (compared to ideal of 1.8)
260/230 ratio: 1.61
pET-Duet-1: 268.0 ng/µL
260/280 ratio: 1.91 (compared to ideal of 1.8)
260/230 ratio: 2.11
Rapid digestion of methylated plasmid DNA using DpnI, in preparation of Gibson Assembly of ATPG into pET-Duet-1:
The nuclease-free water, restriction enzyme 10X buffer, BSA, and Plasmid DNA (the pET-Duet-1 PCR product) was added in the order shown above, then mixed by pipetting. Next, the restriction enzyme (Dpn1) was added to the mixture, and also mixed by pipetting. The resulting mixture was incubated at 37 °C degrees for 15 minutes. The DpnI was then heat inactivated by incubating at 80 °C for 5 minutes, before being placed in the fridge.
Gibson Assembly of ATPG:
pET-Duet-1 (5 µL), ATPG (3.65 µL), and MasterMix (10 µL) was added to nuclease-free water (1.35 µL). The resulting mixtures was incubated at 50 °C for 30 mins. 18 µL of the product was stored at 4 °C for future use. 2 µL was used for transformation, as outlined below.
Transformation:
The chemically competent DH5 α cells (50 µL) were thawed on ice for 30 minutes. The assembled plasmids (2 µL) was then added to these cells, and the tube containing the resulting mixture was flicked gently 5 times. This mixture was then placed on ice for 30 minutes. Following this, the cells were heatshocked at 42 °C for 35 seconds, before being placed on ice for an additional 2 minutes. Next, SOC media (950 µL) was added to the tubes, and the tubes were taped horizontally (to improve aeration, since increased movement of liquid results in more mixing) in a 37 °C incubator shaking at 130 rpm for 1 hour. Following this, 100 microlitres of the cells were spread onto plates with ampicillin. The plates were then incubated overnight at 37 °C.
PCR of NRPS: PCR of NRPS was repeated since the dyed agarose gel (run Monday 19th July) showed too many bands for NRPS under UV light following electrophoresis. In order to hopefully achieve better results this time round, three PCR mixes containing NRPS were created. For each, the volume of NRPS was reduced from 10 µL to 1 µL, and thus the volume of water was adjusted accordingly from 23.5 µL to 32.5 µL. During the PCR reaction, the annealing step was run at a temperature gradient, so that each PCR mix was held at a different temperature (in order to find the optimal practical annealing temperature). Additionally, the denaturation step (in each of the 35 cycles for each PCR mix) was reduced from 1 minute to 30 seconds.
Each PCR mix contained the diluted upstream and downstream primers (5 µL each, 2 µM), dNTP mix (1 µL, 10mM each), nuclease-free water (32.5 µL), NRPS (1 µL, 10 ng), Pfu DNA Polymerase 10X Buffer with MgSO4 (5 µL), Pfu DNA Polymerase (0.5 µL), giving a final volume of 50 µL.
The PCR products were left to soak in the PCR machine at 10 °C overnight.
Admin:
AJ emailed Dr Christopher Moffat from Promega (UK) to thank him for coming to visit our team last Thursday, offering advice about possible career opportunities after graduation, and notifying us about the Student Resource Centre available on the Promega website.
Wiki:
Anna M. made some helpful comments on AJ’s rough draft of the Project Description.
Promotional Video:
With advice from Anna M. and AJ about the sponsorships we have received, Shagun completed the sponsors slide on the Promotional video. Shagun had also added background music, the iGEM 3 second intro video, as well as the credits at the end.
Photo inserts of everyone in the team?
Outreach and Education:
Shagun emailed Vegan Society about a possible collaboration. Vegan Society agreed – so Shagun, AJ, and Yoricka will attend a Teams call with them at 6 pm tomorrow (21st July) in order to plan a possible workshop/event.
Wednesday 21st July
In the laboratory:
21/07/21 - Anna, Maia, Julia, AJ and Yoricka
NRPS was taken out of the PCR machine (held at 10 °C overnight) and stored in the fridge.
The bacterial colonies (containing pET-Duet-1 plasmids assembled with ATPG) grown overnight were taken out of the 37 °C incubator just before lunch. These colonies were placed in the fridge.
PCR of O-MT
PCR of O-MT was repeated since the agarose gel did not show a clear enough band under UV light (when viewed on Monday 19th July). Thus, three new PCR mixes containing O-MT were created: these were placed at different points along a temperature gradient (centred at 63 °C, with a 12 °C range) during the annealing stage of each PCR cycle. The volume of O-MT in each PCR mix was reduced from 10 µL to 1 µL (thus the volume of water in each mix was adjusted from 23.5 µL to 32.5 µL). The denaturation step (in each of the 35 cycles) was also reduced from 1 minute to 30 seconds.
Each PCR mix contained the diluted upstream and downstream primers (5 µL of each primer, 2 µM), dNTP mix (1 µL, 10mM each), nuclease-free water (32.5 µL), O-MT (1 µL, 10 ng/50 ul PCR reaction), Pfu DNA Polymerase 10X Buffer with MgSO4 (5 µL), Pfu DNA Polymerase (0.5 µL), giving a final volume of 50 µL.
2% agarose gel electrophoresis
The DNA ladders (100 bp DNA ladder (100-1000bp in size), and the Quick-load Purple 1 kb Plus DNA ladder (1000bp- 10kb in size), and the dye (Blue/Orange 6X loading dye) were defrosted.
In one eppendorf tube, the small DNA ladder (100-1,000 bp in size) (5 µL) was added to the Blue/Orange Dye (1 µL). For the DHQS A, DHQS B, DHQS C, NRPS B, NRPS C, O-MT A, O-MT B, and O-MT C PCR gene products, each PCR gene product (1 µL) was added to Blue/Orange Dye (1 µL) and nuclease-free water (4 µL). The resulting mixtures were added to the wells of the 2% agarose gel in the order specified below.
A small mistake ensued with the NRPS A PCR gene product: 5 µL of the gene product was added to Blue/Orange dye (1 µL). To rectify this, the total mixture (6 µL) was added back to the complete PCR NRPS A gene product (now 51 µL), so that 50 µL of PCR NRPS A gene product ended up being diluted with Blue/Orange dye (1 µL). This NRPS A PCR gene product mixture (1 µL) was added to Blue/Orange dye (1 µL) and nuclease-free water (4 µL). The resulting mixture (6 µL) was added to the 3rd well of the 2% agarose gel.
The consequences of this mistake are that the PCR NRPS A gene product is slightly less concentrated than initially hoped for in this mixture, while the Blue/Orange dye is present in a slightly higher concentration than initially planned. This may affect the visualisation of the bands under UV light, although this is unlikely. With regards to Gibson Assembly, the dye present in the complete NRPS A PCR gene product mixture will be removed during the clean-up procedure, hence the insertion of NRPS into pET-Duet-1 should not be affected.
The already-dyed large (1,000 – 10 kb in size) DNA ladder (5 µL) was added to the 15th well in the 2% agarose gel.
Once all the PCR DNA products had been inserted into the wells, the electrophoresis tank was run at 80 V for 1 hour, with the 2% agarose gel submerged in 1X TAE buffer. Growing the colonies overnight: LB broth (10 mL) was added to ampicillin (10 µL, 100mg/mL). Then one large colony of DH5α bacteria (containing the ATPG-assembled pET-Duet-1 plasmid) was added to this mixture. This entire procedure was repeated for 2 additional large DH5α bacterial colonies and 1 small bacterial colony. Then a control was set up, where LB broth (10 mL) was added to ampicillin (10 µL, 100mg/mL), while no bacteria was added. These colonies were incubated overnight at 37 °C alongside the control, shaking at 160 rpm.
The circled colonies are the ones grown overnight. The three circles next to/below the label correspond to the large bacterial colonies, while the small bacterial colony we took is indicated by the circle above the label.
Dyeing the 2% agarose gel:
Nucleic acid dye (15 µL) was added to 1X buffer TAE (150 mL). This was then poured over the 2% agarose gel, and rocked for 30 minutes.
Making up 1% agarose gels.
A 0.8 % agarose solution was already made up (whereby agarose (1.2 g) had been dissolved in 1X TAE (150 mL)). Agarose (0.4 g) was added to make the concentration up to 1%.
Technically, addition of agarose (0.3 g) would have made the concentration up to 1%, but 0.4 g was used to make up for the agarose residue lost upon transfer between glassware.
The resulting mixture was microwaved (being swirled at 30 second intervals) until the agarose dissolved. The solution was left to cool, and then it was poured into the gel rig. Once the solution had set, the agarose gel was placed in the fridge.
The gel bands should have been at 100 and 500.
NRPS – 2,667
O-MT – 840
DHQS - ~1,500
It looks like we have products of expected size, as well as some non-specific stuff. In each case, it looks like the product is the predominant ‘species’ or present in the highest amount.
Of the 3 reactions run, the first two O-MTs are able to be run by Gibson Assembly.
If we had loaded only half the amount onto the gel, we may not have seen the non-specific products. So this may have been a loading issue.
Don't run NRPS, since our wanted product does not even make up 50% of the product total. With DHQs you can be optimistic – you can use the middle band for Gibson assembly.
We have improved on the specificity since the first occasion on which we ran the PCR gene products on the gel.
You’d expect, that as the temp increases, you see less of the non-specific products. This is NOT REALLY what we see in our results following a gradient of the annealing temperature.
We can either reduce the amount of the template present
3 in the middle (DHQS have improved in specificity)
3 on the right (O-MT) is an improvement, since in the previous attempt, the bands were very un-specific.
We have definitely amplified the product of interest, looking at the sizes of the bands.
We can either: try processing two of these
Or we can run everything out on a gel, cut out the band of interest
PCR mix (remaining 49 microlitres can be used as a template) and repeat the PCR>
Thanogen Part difficulties
The IDT website was used to construct gblock fragments for the Thanogen composite parts. Unfortunately, when the sequences were inserted into the order and checked with difficulty scores the parts had many manufacturing difficulties as shown below) which meant the scores were far too high and IDT would not manufacture them for us. The plan is to look into editing these sequences to try and get them to an acceptable manufacturing score for future teams to use.
Whole-Team meeting Notes:
With this Friday (23rd July) being the check-in day for Roster and Track, our PI (Dr Chris Hooley) added us to two tracks: our first choice being the Environmental Track, and our second choice being the Open Track. Dr Chris Hooley also emailed our other PI (Dr Jacqueline Nairn) about setting up an iGEM account, so that he could add her onto the Roster (everyone else on the team is on the Roster).
Shagun notified AJ that she needs to sign a consent form, in order to approve her voice being used in the audio of the video. AJ then signed the consent form.
AJ said that Twist Bioscience hadn’t supplied us with any DNA during our project yet, so we can remove the Twist Bioscience logo from our Promotional video.
Shagun said that IDT hadn’t reached out to us to supply their logo, so Anna M. said that, if IDT don’t get back to us in time, we can simply write their company name at the bottom of our sponsorship slide in the video instead of using their Logo.
Shagun notified the team that today was the last day we could give feedback on the promotional video (since it is due on Friday). The team collectively decided that it would be best not to include photos of each team member in the video, since iGEM mentioned that the credits should be written in text (we believe this means that photos should not be included in this section), and also because the frame where we conceptually introduce the iGEM team looks cluttered if we add each our photographs in.
AJ, Yoricka, Anna M., Chloe, and Shagun will attend the VegSoc meeting tonight, to discuss a potential collaboration.
Maia also mentioned that IDT would not attempt to produce the composite parts created by last year’s iGEM team. Dr Chris Hooley said that this might prove to be a good opportunity for us – we could spend some time changing these composite parts to make them more manufacturable - thus working towards completing the ‘Engineering success’ or ‘Improvement of an existing part’ medal criteria.
VegSoc Meeting Notes
Shagun, Yoricka, Chloe, and AJ met Heidi Bough (a committee member from Vegsoc).
We collectively agreed that iGEM should present information in the form of an interactive workshop, since this is likely to engage more members.
AJ mentioned that, during the workshop, the iGEM team would likely present some facts and statistics about the damage that many sunscreens available on the market, then talk a little bit about our project (promoting our work to members, hopefully getting their opinion on whether they would use the sunscreen), and lastly present some sustainable and affordable sunscreen alternatives that members could buy instead of harmful sunscreens.
Heidi Bough mentioned that she is also a part of Marine society – she advocated that Marine Society would also be a good collaboration option with regards to our project.
Shagun will send an email to Marine Society about a potential collaboration (this can either take place in conjunction with the VegSoc x iGEM workshop event, or it could be a separate event). Heidi Bough will send a message in the Marine Society group chat to notify members of this email + promote the idea of a collaboration.
In order to promote both the use of sustainable sunscreens, as well as joining iGEM or VegSoc, we thought it would be a good idea to host the workshop at the beginning of next semester – either in Fresher’s week or Week 1, where engagement amongst potential new members might be highest.
Last year, many of VegSoc’s events were putlock/free dinners – we considered the possibility of conducting our workshop in an event of this format, and thought it might achieve higher engagement.
We also considered Covid restrictions, and decided that doing an outdoor event would be better than an indoor event, since the requirement for social distancing indoors might make the event less engaging.
Future actions: plan a call with Marine Society (if/when they get back to us) to see what type of event they’d like to host with us (I.e. will this be the same collaborative event with VegSoc, or a different event?). Once we have confirmation of the types of events we’ll be planning, some members of the iGEM team can start creating educational/workshop materials. Once this is finished, we can present these PowerPoints/presentations/workshops to VegSoc (and potentially Marine Society) to collect their feedback on our planned workshop, and further plan the real events. Following this, members of our iGEM team can create promotional material for this event, and advertise this on social media (and share it with VegSoc + Marine Society so they can advertise the event too!).
Thursday 22nd July
In the laboratory:
22/07/21 - Anna, Julia, AJ, Yoricka, and Maia
Miniprep the assembled plasmids
The DH5α bacterial colonies grown overnight were taken out of the incubator and centrifuged at 1,500 x g for 10 minutes. The LB broth was then discarded (the falcon tubes were held upside town on a paper towel to dry the pellets), PBS (500 µL) was added to the pellets, and then the resulting mixture was vortexed until the pellets were resuspended. The mixtures were microcentrifuged again at maximum speed for 15 seconds, and the PBS discarded. Cell lysis buffer (200 µL) was added to each of the pellets (the protocol recommended adding 100 µL, but during our previous miniprep procedure we found that this volume wasn’t enough since the resulting mixture turned very sticky). The resulting solutions were mixed by inverting 6 times, flicked, and then mixed by pipetting until the pellets were resuspended. Neutralisation solution (350 µL) was added to each mixture, and then the resulting mixtures were also inverted and flicked until they turned completely yellow. These mixtures were then microcentrifuged at maximum speed for 3 minutes. The supernatant was pipetted into a minicolumn and then microcentrifuged at maximum speed for 15 seconds. The flowthough was discarded, the Endotoxin Removal Wash (200 µL) was added to minicolumn, and then the minicolumn was centrifuged at maximum speed for 15 seconds again. Column Wash Solution (400 µL) was then added to the minicolumn, before centrifuging at maximum speed for 30 seconds. The minicolumn was then transferred to a clean eppendorf tube, then Elution Buffer (30 µL) was added directly to the minicolumn matrix. This was left to stand at room temperature for 1 minute. To elute the plasmid DNA, the eppendorf tube was microcentrifuged at maximum speed for 15 seconds. Any miniprep not used in the restriction digest was placed in the freezer.
Restriction digest
The Restriction Enzyme 10X Buffer D1(2 µL), nuclease-free water (15 µL), and finally NcoI Restriction Enzyme (1 µL, 10 µ/µL) was added to the miniprep product (2 µL). The resulting mixture was placed in the incubator at 37 °C for 2 hours.
Running the gel
Loading dye (4 µL) was added to each of the digests (20 µL). Loading dye (1 µL) was also added to the small 100 bp ladder (5 µL). The large ladder (5 µL) was already pre-dyed.
Making a 1.5 % agarose gel (1.5% gives better separation between the 1 kb – 5 kb range).
Agarose (1.125 g) was added to 1X TAE buffer (75 mL). The resulting mixture was microwaved (being swirled at 30 second intervals) until the agarose dissolved. The solution was left to cool, and then it was poured into the gel rig. The gel rig was taped to make larger wells. Once the solution had set, the agarose gel was placed in the fridge.
Dyeing the 1.5% agarose gel: (this gel will need to be run for 1.5 hours). Nucleic acid dye (15 µL) was added to 1X buffer TAE (150 mL). This was then poured over the 2% agarose gel, and rocked for 30 minutes.
Mega Wells GEL
All look the same
800 bp is there - just can’t be seen properly (not a lot of it – the digest was not very effective). Maybe there is too much DNA (unlikely?), or maybe the NcoI is dodgy?
Top two bands – top band is bang on 6 kb – looks like the fragment from the digested, assembled plasmid. The lower top band is running at ~4.5 kb. We are not expecting a fragment of this size – is there another NcoI site? This is likely to be the original plasmid that is digested, this is not a linear molecule – it is supercoiled, so it will run through the gel easier – so it looks smaller in bp size than it actually is.
3 fragments – 2 of 3 fragments are the size we expect. One fragment is not – must be the intact, supercoiled plasmid.
4/4 - all of them worked!!!!
Choose 2 to sequence from (A and B) - sequencing is expensive
We need to reimburse – using SeaChangers
The minipreps can stay frozen
Get 4 barcodes (forwards and reverse for each digest) from Val tomorrow
Online associate barcode with articular sequencing reaction
Quantify what the two (A and B) are using the nanodrop
Tomorrow they can go in the GATC box
Quantified pET-Duet-1 plasmids assembled with ATPG, A and B, using the NanoDrop Spectrophotometer:
Results:
Restriction digest A: 302.8 ng/µL - decent amount
260/280 ratio: 1.91 (compared to ideal of 1.8)
260/230 ratio: 1.69
Restriction digest B: 205.3 ng/µL
260/280 ratio: 1.91 (compared to ideal of 1.8)
260/230 ratio: 1.92
NcoI - will produce product fragment lengths of 803 bp and 5995 bp in assembled plasmid.
In the original plasmid, it would cut once – size would be 5420 bp.
Instead of performing multiple PCRs that might never improve, we planned to make bigger wells – by applying autoclave tape.
Then run them all on a gel and cut out the appropriate bands (gel extraction - use Promega kit). Assuming the sizes are correct, we can carry out gel extraction, purify this, quantify, use this in gibson assembly.
Quantify, and use these gene fragments in Gibson Assembly.
Friday 23rd July
In the laboratory:
23/07/21 - Anna, Julia, AJ, Yoricka. Sent off pET-Duet-1 plasmids assembled with ATPG (clones A and B) for sequencing
pET-Duet-1 ATPG clone A (4 µL, 302.8 ng/µL) was added to nuclease-free water (11 µL), giving a more diluted concentration of pET-Duet-1 ATPG clone A (15 µL, 80 ng/µL). pET-Duet-1 ATPG clone B (6 µL, 205.3 ng/µL) was also added to nuclease-free water (9 µL), again giving a diluted concentration of pET-Duet-1 ATPG clone B (15 µL, 80 ng/µL). Both the assembled clones were sent to Eurofins Genomics GATC Services for Custom DNA sequencing: each clone was sequenced forwards and backwards, with petup being the forwards primer, and DuetDown1 being the reverse primer.
Barcodes: 2HF368 A: forwards 2HF369 B: reverse
2HF270 A: rev
Ran a DNA Electrophoresis of NRPS, DHQS, and O-MT PCR products on the 1.5 % agarose gel
NRPS PCR samples A (49 µL), B (49 µL), and C (49 µL) were mixed together, and added to loading dye (30 µL). DHQS PCR samples A (49 µL), B (49 µL), and C (49 µL) were mixed together, and also added to loading dye (30 µL). O-MT PCR samples A (49 µL) and B (49 µL) were mixed together, and added to loading dye (17 µL). The resulting NRPS (177 µL), DHQS (177 µL), and O-MT (115 µL) PCR solutions, as well as the large DNA ladder (5 µL) were added to the wells of the gel in the following order:
1st well: large ladder (5 µL)
2nd well: N/A
3rd-5th wells (inclusive): O-MT (98 µL) + Loading dye (17 µL)
6th well: N/A
7th-10th wells (inclusive): NRPS (147 µL) + Loading dye (30 µL)
11th well: N/A
12th -15th wells (inclusive): DHQS (147 µL) + Loading dye (30 µL)
16th well: big ladder (5 µL)
The electrophoresis pack was run in 1X TAE at 80 V for 1 hour. *Mistake – we should have run this for 90 minutes to get better separation.
Dyeing the 1.5% agarose gel
Diamond Nucleic acid dye (15 µL) was added to 1X TAE buffer (150 mL). This was then poured over the 1.5% agarose gel and rocked for 30 minutes.
The large ladders on either side of the gel were not visible. NRPS, DHQS, and O-MT PCR products didn’t work either because the large wells (formed by tape) were too deep. Because leakage took place as well, we wouldn’t have been able to carry out gel extraction. Maybe the noise is making it so that you can’t see the ladder.
Later we discovered that the dye inside the ladder was concentrated at the bottom of the tube – this may have been why our ladders didn’t show up clearly under UV light.
Gel extractions:
Heating block to be hot – for the extractions
Starting again:
Choose temperatures which we’ll be using
Run O-MT PCR again
One Monday morning, one Monday afternoon, one at night – we'll have 3 done on Tuesday.
Mega wells again on Tuesday
Pick temperature that you think is going to do best, but have multiple replicates, which you can combine to the megawells.
PCR of DHQS
In preparation for the PCR of DHQS (1234 bp), the working stock of each primer (4 µL, 10 µL) was diluted by a factor of 5 using nuclease free water (16 µL). Three lots of PCR of DHQS were then conducted. The PCR mix contained the now-diluted upstream and downstream primers (5 µL each, 2 µM), dNTP mix (1 µL, 10mM each), nuclease-free water (32.5 µL), DHQS (1 µL, 10 ng/50 ul PCR reaction) Pfu DNA Polymerase 10 X Buffer with MgSO4 (5 µL), Pfu DNA Polymerase (0.5 µL), giving a final volume of 50 µL.
Saturday 24th July
Continued to work on the project, and troubleshoot wetlab issues.
Sunday 25th July
Continued team work on the project.
Week 8:
Monday 26th July
In the laboratory:
26/07/21 - Anna, Maia, Julia, AJ, Yoricka.
PCR of O-MT
In preparation for the PCR of O-MT (840 bp), the working stock of each primer (4 µL, 10 µL) was diluted by a factor of 5 using nuclease free water (16 µL). Three lots of PCRs of O-MT were then conducted. The PCR mix contained diluted upstream and downstream primers (5 µL each, 2 µM), dNTP mix (1 µL, 10mM each), nuclease-free water (32.5 µL), O-MT (1 µL, 10 ng/50ul PCR reaction), Pfu DNA Polymerase 10 X Buffer with MgSO4 (5 µL), Pfu DNA Polymerase (0.5 µL), giving a final volume of 50 µL.
PCR of NRPS
In preparation for the PCR of NRPS ( bp), the working stock of each primer (4 µL, 10 µL) was diluted by a factor of 5 using nuclease free water (16 µL). Three lots of PCR of NRPS were then conducted. The PCR mix contained the now-diluted upstream and downstream primers (5 µL each, 2 µM), dNTP mix (1 µL, 10mM each), nuclease-free water (32.5 µL), NRPS (1 µL, 10ng/50 ul PCR reaction), Pfu DNA Polymerase 10 X Buffer with MgSO4 (5 µL), Pfu DNA Polymerase (0.5 µL), giving a final volume of 50 µL. The programmed temperatures and durations of each step are detailed below.
About 30 minutes into the PCR, the PCR machine stopped working and came up with an error code (error code 1013, also code 1025, and 1007). The lab technician stated that this was an electrical issue, and he started the PCR again from the start.
Making 1.5% agarose gels
Agarose (2.25 g) was added to 1X TAE buffer (150 mL). The resulting mixture was microwaved (swirled at 30 second intervals) until the agarose dissolved. The solution was left to cool, and then a portion of it was poured into a gel rig. This gel rig had mega wells (whereby there were 3 mega-wells made up of 4 normal-sized wells each, as well as there being two normal-sized wells on either side for the ladders). Once the solution had set in the rig, the agarose gel was placed in the fridge. The comb was risen up/adjusted here.
Analysing the sequencing information obtained from Eurofins Genomics
Certain position where it is a GAG in the A sequence – in the coding sequence of the gene.
In clone B, at the same position of the coding sequence of the gene, it’s a TAG – it has created a mistake – a premature stop codon in the gene – so we have a truncated protein.
Shirley thinks that (a chance of a mistake/mutation occurring either In E.coli or the PCR is very low). SO, we think that when the gene was originally made (when they made a synthesis of DNA) they put in a wrong nucleotide. Occasionally, there is a mixed population.
Good lesson for future teams
Our results show that not every seqeuncing reaction works successfully, and that mistakes can be made in oligo synthesis.
Quantifying the pRSF PCR products using the NanoDrop spectrophotometer.
Results:
pRSF clone A: 29.1 ng/µL
260/280 ratio: 1.76 (compared to ideal of 1.8)
260/230 ratio: 1.88
pRSF clone B: 82.9 ng/µL
260/280 ratio: 1.86 (compared to ideal of 1.8)
260/230 ratio: 0.94 (likely due to presence of guanidine thiocyanate, which was present in the PCR cleanup).
Tuesday 27th July
In the laboratory:
27/07/21 - Anna, Maia, Julia, AJ, Yoricka.
PCR of clone A of pET-Duet-1 with ATPG insert
The ATPG pET-Duet-1 clone A PCR products (1 µL, 302 ng/µL) were added to nuclease-free water (999 µL). Then the forwards and reverse primers (1 µL each) were added to the now-diluted DNA (8.28 µL, 0.302 ng/µL). Next, Nuclease-free water (4.72 µL) and MasterMix (25 µL) were added, and the resulting mixture (50 µL) inverted to mix. This procedure was repeated two more times.
1.5 % agarose gel electrophoresis of O-MT, DHQS, and NRPS PCR products (Mega Well Gel #2)
1X TAE buffer was poured into the gel rig until it covered the 1.5 % agarose gel. At the same time, the Quick-load Purple 1 kb Plus DNA ladder (100bp- 10kb in size), and the dye (Blue/Orange 6X loading dye) were defrosted.
The three DHQS PCR replicates (50 µL each) were added to the Blue/Orange 6X loading dye (30 µL), and the resulting mixture was mixed by flicking. This mixture (100 µL) was then added to the first mega well in the agarose gel. This procedure was repeated for the NRPS and O-MT PCR products.
The already-dyed large (1,000 – 10 kb in size) DNA ladder (5 µL) was then added to the 1st and 16th wells in the agarose gel.
The PCR gene products and ladders were placed in the following order in the gel:
1st well: large ladder
2nd-5th wells: DHQS
6th well: blank
7th - 10th wells: NRPS
11th well: blank
12th-15th wells: O-MT
16th well: large ladder
The electrophoresis tank was left to run at 80V for 90 minutes.
Dyeing the 1.5% Agarose Gel
10X TAE buffer (101.5 mL) was added to water (915 mL). Nucleic acid dye (15 µL) was then added to the resultant 1X buffer TAE (150 mL). This was then poured over the 1.5% agarose gel, and rocked for 30 minutes.
Gel Extraction
The bands containing the O-MT, NRPQ, and DHQS genes were excised from the gel and stored in the fridge.
Gel Slice Preparation
Weighing the masses of each gel slice:
DHQS: 556 mg
NRPS: 391 mg
O-MT: 417 mg
Membrane binding solution (556 µL, 391 µL, and 417 µL) was added to the DHQS, NRPS, and O-MT gel slices respectively, in a ratio of 1 µL of Membrane Binding Solution per 1 mg of gel slice. The resulting mixtures were vortexed, then incubated at 62 °C for 2 hours. After this time, they still hadn’t melted, so we will repeat this step tomorrow.
The nuclease-free water, restriction enzyme 10X buffer B, BSA, and Plasmid DNA (the pRSFDuet-1 PCR product B) was added in the order shown above, then mixed by flicking. Next, the restriction enzyme was added to the mixture, and also mixed by flicking. The resulting mixture was incubated at 37 °C degrees for 15 minutes. The DpnI was then heat inactivated by incubating at 80 °C for 5 minutes, before being placed in the fridge.
Wednesday 28th July
In the laboratory:
28/07/21 - Anna, Maia, Julia, AJ, Yoricka.
Gel slice preparation
The three gel slices (containing DHQS, NRPS, and O-MT respectively) were heated up to 60 °C for 3 hours. The gel slices didn’t melt further. It was then discovered that yesterday, Membrane Wash solution was added to each gel slice instead of Membrane Binding solution. Now, membrane binding solution (391 µL, and 417 µL) was added to the NRPS, and O-MT gel slices respectively. Membrane binding solution (~500 µL) was added to DHQS (556 µL wouldn’t fit into the tube). The O-MT, NRPS, and DHQS gel slices were incubated at 60 °C for 10 minutes, at which point they all (apart from DHQS) had dissolved.
To bind the DNA, the dissolved gel mixture was poured into a minicolumn assembly, then kept at room temperature for 1 minute. This was centrifuged at 16,000 x g for 1 minute, then the flowthrough was discarded. (ethanolic) Membrane Binding solution (55 µL) was then added to the DHQS product again, since some gel slice was remaining in the tube. This was incubated at 60 °C for 3 minutes until the gel had dissolved. The dissolved gel mixture was poured into a minicolumn assembly again, kept at room temperature for 1 minute, centrifuged at 16,000 x g for 1 minute, and the flowthrough discarded. Next, (ethanolic) Membrane Wash Solution (700 µL) was added to each of the DHQS, NRPS, and O-MT products, and the minicolumn assembly centrifuged at 16,000 x g for 1 minute. The flowthrough was discarded, then (ethanolic) Membrane Wash Solution (500 µL) was added to each of the DHQS, NRPS, and O-MT products again, and the minicolumn assembly centrifuged at 16,000 x g for 5 minutes. The flowthrough was discarded, and the column assembly was centrifuged at 16,000 x g for 1 minute.
The minicolumn was then transferred to a 1.5 microcentrifuge tube. Nuclease-free water (50 µL) was added, the tube incubateed at room temperature for 1 minute, then centrifuged at 16,000 x g for 1 minute. The DNA was then quantified using a NanoDrop Spectrophotometer.
Quantifying DHQS, NRPS, and O-MT using the NanoDrop spectrophotometer
DHQS: 9.7 ng/µL
260/280 ratio: 1.29 (compared to ideal of 1.8)
260/230 ratio: 0.03
This has the weakest concentration and 260/280 ratio.
NRPS: 26.3 ng/µL
260/280 ratio: 1.56 (compared to ideal of 1.8)
260/230 ratio: 0.20
O-MT: 14.2 ng/µL
260/280 ratio: 1.76 (compared to ideal of 1.8)
260/230 ratio: 0.17
Clean-up of the three PCR products of ATPG-containing pET-Duet-1 plasmid DNA:
The three PCR amplifications were added to three minicolumn assemblies. Immediately after, Membrane Binding solution (50 µL) was added to each minicolumn assembly (this is not what we should have done – we should have added the Membrane Binding Solution to the three PCR amplifications, before transferring the prepared PCR product into the minicolumn assembly). The minicolumn assemblies were incubated at room temperature for 1 minute, before being centrifuged at 16,000 x g for 1 minute. The flowthrough was discarded, then ethanolic Membrane Wash Solution (700 µL) was added, the minicolumn assembly centrifuged at 16,000 x g for 1 minute, and the flow-through discarded. (Ethanolic) Membrane Wash Solution (500 µL) was added, the minicolumn assembly centrifuged at 16,000 x g for 5 minutes, and the flow-through discarded. The empty column assembly was then centrifuged at 16,000 x g for 1 minute. The minicolumn was placed inside a clean Eppendorf tube, Nuclease-free water (50 µL) was then added to the minicolumn, the minicolumn was incubated at room temperature for 1 minute, then centrifuged at 16,000 x g for 1 minute. The ATPG-containing pET-Duet-1 plasmid PCR products were then quantified.
Quantifying the ATPG-containing pET-Duet-1 plasmid PCR products 1-3 using the NanoDrop spectrophotometer
PCR 1: 37.8 ng/µL
260/280 ratio: 1.77 (compared to an ideal of 1.8)
260/230 ratio: 0.45
PCR 2: 44.5 ng/µL
260/280 ratio: 1.67 (compared to ideal of 1.8)
260/230 ratio: 0.40
PCR 3: 59.4 ng/µL
260/280 ratio: 1.78 (compared to ideal of 1.8)
260/230 ratio: 0.73
We will the products of PCR 3 for digestion since these yield the highest concentration, as well as the best 260/280 ratio.
The pET-Duet-1 ATPG products of PCR 1 and 2 were placed in the fridge.
The nuclease-free water, restriction enzyme 10X buffer B, BSA, and Plasmid DNA (the ATPG-inserted pET-Duet-1 plasmid products of PCR 3) was added in the order shown above, then mixed by flicking. Next, the restriction enzyme was added to the mixture, and also mixed by flicking. The resulting mixture was incubated at 37 °C degrees for 15 minutes. The DpnI was then heat inactivated by incubating at 80 °C for 2 minutes.
Gibson Assembly of NRPS into ATPG-containing pET-Duet-1
NRPS (2.23 µL), pET-Duet-1 ATPG (5 µL), and nuclease-free water (2.77 µL) was added to the MasterMix (10 µL). The resulting mixture was incubated at 50 °C for 30 mins, then cooled on ice. 18 µL of the product was stored at 4 °C for future use. 2 µL was used for transformation, as outlined below.
Gibson Assembly of DHQS into pRSF-Duet
DHQS (5 µL) and pRSF-Duet (5 µL) was added to MasterMix (10 µL). The resulting mixture was incubated at 50 °C for 30 mins, then cooled on ice. 18 µL of the product (20 µL) was stored at 4 °C for future use. 2 µL was used for transformation, as outlined below.
Transformation of pET-Duet-1 plasmid assembled with ATPG and NRPS:
The chemically competent DH5α cells (50 µL) were thawed on ice for 5 minutes. The assembled pET-Duet-1 plasmids (2 µL) were then added to these cells, and the tube containing the resulting mixture was flicked gently 5 times. The mixture was then placed on ice for 30 minutes. Following this, the cells were heat shocked at 42 °C for 35 seconds, before being placed on ice for an additional 2 minutes. Next, SOC media (950 µL) was added to the tubes, and the tubes were taped horizontally (to improve aeration, since increased movement of liquid results in more mixing) in a 37 °C incubator shaking at 130 rpm for 1 hour. Following this, 100 microlitres of the cells were spread onto plates with ampicillin. This was repeated for another plate, then the plates were then incubated overnight at 37 °C.
Transformation of pRSF-Duet-1 plasmid assembled with DHQS:
The chemically competent DH5α cells (50 µL) were thawed on ice for 5 minutes. The assembled pRSF-Duet-1 plasmids (2 µL) were then added to these cells, and the tube containing the resulting mixture was flicked gently 5 times. The mixture were then placed on ice for 30 minutes. Following this, the cells were heat shocked at 42 °C for 35 seconds, before being placed on ice for an additional 2 minutes. Next, SOC media (950 µL) was added to the tubes, and the tubes were taped horizontally (to improve aeration, since increased movement of liquid results in more mixing) in a 37 °C incubator shaking at 130 rpm for 1 hour. Following this, 100 microlitres of the cells were spread onto a plate with kanamycin. This was repeated for another plate, then the plates were then incubated overnight at 37 °C.
Thursday 29th July
In the laboratory:
29/07/21 - Anna, Maia, Julia, AJ, Yoricka.
Picking Colonies, and Overnight Growth
In the morning, the four plates incubated overnight were observed. Each plate had colonies growing on them, but these were fairly small, so the plates were left to grow for a few more hours. The plates were then transferred to the fridge.
PET-Duet-1 ATPG/NRPS 1A (Plate 1, Colony A) - Ampicillin resistance
PET-Duet-1 ATPG/NRPS 1B (Plate 1, Colony B) - Ampicillin resistance
PET-Duet-1 ATPG/NRPS 2C (Plate 2, Colony C) - Ampicillin resistance
PET-Duet-1 ATPG/NRPS 2D (Plate 2, Colony D) - Ampicillin resistance
PRSF-Duet-1/DHQS 1A (Plate 1, Colony A) - Kanamycin resistance
PRSF-Duet-1/DHQS 1B (Plate 1, Colony B) - Kanamycin resistance
PRSF-Duet-1/DHQS 2A (Plate 2, colony C) - Kanamycin resistance
PRSF-Duet-1/DHQS 2B (Plate 2, colony D) - Kanamycin resistance
Kanamycin Control
Ampicillin Control
For both pET-Duet-1, and pRSF-Duet-1, 3 big colonies (A, B, and C) were grown, as well as a small colony (D).
LB broth (10 mL) was added to ampicillin (10 µL, 100mg/mL). Then, from plate 1, one large colony of DH5α bacteria (containing the ATPG/NRPS-assembled pET-Duet-1 plasmid) was added to this mixture. This entire procedure was repeated for 2 additional large DH5α bacterial colonies (one taken from plate 1, the other from plate 2) and 1 small bacterial colony (taken from plate 2). Then a control was set up, where LB broth (10 mL) was added to ampicillin (10 µL, 100mg/mL), while no bacteria was added. These colonies were incubated overnight at 37 °C alongside the control, shaking at 160 rpm.
LB broth (10 mL) was then added to kanamycin (10 µL, 50mg/mL). From plate 1, one large colony of DH5α bacteria (containing the DHQS-assembled pRSF-Duet-1 plasmid) was added to this mixture. This entire procedure was repeated for 2 additional large DH5α bacterial colonies (one taken from plate 1, the other from plate 2) and 1 small bacterial colony (taken from plate 2). Then a control was set up, where LB broth (10 mL) was added to kanamycin (10 µL, 50mg/mL), while no bacteria was added. These colonies were incubated overnight at 37 °C alongside the control, shaking at 160 rpm.
The circled colonies are the ones grown overnight. The three circles next to/below the label correspond to the large bacterial colonies, while the small bacterial colony we took is indicated by the circle above the label.
Protein structure prediction - Maia and Julia
Blastx was used to convert the DNA sequences for each gene into protein sequences. HHpred was then used to convert these sequences into predicted secondary structures. PyMol was also used to visualize and orientate these structures, probability plots were also recorded (see protein predictions document).
Blastx and Protein Database Bank (PDB) were used to search for existing protein structures of these genes to allow future comparison after Circular dichroism spectroscopy. On PDB, we were able to find existing protein structures for NRPS, O-MT, and DHQS. Also for DHQS and NRPS, there were existing structures that matched the protein code of the theoretical models produced by PyMol (use image from Known Protein Structures doc).
Friday 30th July
In the laboratory:
30/07/21 - Anna, Julia, AJ, Yoricka.
Miniprep the assembled plasmids
The DH5α bacterial colonies grown overnight were taken out of the incubator and centrifuged at 15,000 x g for 10 minutes. PET-Duet-1 colonies 1A, 1B, and 2C were transparent following centrifugation, while pET-Duet-1 colony 2D was still slightly cloudy after centrifugation. All the pRSF colonies were transparent following centrifugation. The LB broth was then discarded from each tube (the falcon tubes were held upside town on a paper towel to dry the pellets), PBS (500 µL) was added to each pellet, and then the resulting mixture was vortexed until each pellet was resuspended. Each mixture was microcentrifuged again at maximum speed for 15 seconds, and the PBS discarded. Cell lysis buffer (200 µL) was added to each of the pellets (the protocol recommended adding 100 µL, but during our previous miniprep procedure we found that this volume wasn’t enough since the resulting mixture turned very sticky). The resulting solutions were mixed by inverting 6 times, flicked, and then mixed by pipetting until the pellets were resuspended. Neutralisation solution (350 µL) was added to each mixture, and then the resulting mixtures were also inverted and flicked until they turned completely yellow. These mixtures were then microcentrifuged at maximum speed for 3 minutes. The supernatant was pipetted into minicolumns and then each minicolumn was microcentrifuged at maximum speed for 15 seconds. The flowthough was discarded, the Endotoxin Removal Wash (200 µL) was added to each minicolumn, and then the minicolumns were centrifuged at maximum speed for 15 seconds again. Column Wash Solution (400 µL) was then added to each minicolumn, before centrifuging at maximum speed for 30 seconds. The minicolumns were then transferred to a clean eppendorf tube, then Elution Buffer (30 µL) was added directly to each minicolumn matrix. These matrices were left to stand at room temperature for 1 minute. To elute the plasmid DNA, the eppendorf tubes were microcentrifuged at maximum speed for 15 seconds. Any miniprep not used in the restriction digest was placed in the freezer.
Restriction digest - Assembled pET-Duet-1 plasmids:
The Restriction Enzyme 10X Buffer D2 (2 µL), nuclease-free water (15 µL), and finally the MluI Restriction Enzyme (1 µL, 10 µ/µL) was added to the miniprep product (2 µL). This was repeated for each of the assembled pET-Duet-1 miniprep products. The four resulting mixtures were placed in the incubator at 37 °C for 2 hours.
Restriction digest - Assembled pRSF-Duet-1 plasmids:
The Restriction Enzyme 10X Buffer E3 (2 µL), nuclease-free water (15 µL), and finally the HindIII Restriction Enzyme (1 µL, 10 µ/µL) was added to the miniprep product (2 µL). This was repeated for each of the assembled pRSF-Duet-1 miniprep products. The four resulting mixtures were placed in the incubator at 37 °C for 2 hours.
Running the gel
Loading dye (4µL) was added to each of the digests (20 µL). Loading dye (1 µL) was also added to the small 100 bp ladder (5 µL). The large ladder (5 µL) was already pre-dyed.
Then the dyed restriction digests (15 µL), the dyed small 100 bp ladder (6 µL), and the large 10 kb ladder (5 µL) were added to the 1% agarose gel (submerged in 1X TAE buffer) in the following order:
1st well: large (10 kb) ladder
2nd well: N/A
3rd well: pET-Duet-1 ATPG/NRPS plasmid digest product, colony A
4th well: pET-Duet-1 ATPG/NRPS plasmid digest product, colony B
5th well: pET-Duet-1 ATPG/NRPS plasmid digest product, colony C
6th well: pET-Duet-1 ATPG/NRPS plasmid digest product, colony D
7th well: N/A
8th well: pRSF-Duet-1 DHQS plasmid digest product, colony A
9th well: pRSF-Duet-1 DHQS plasmid digest product, colony B
10th well: pRSF-Duet-1 DHQS plasmid digest product, colony C
11th well: pRSF-Duet-1 DHQS plasmid digest product, colony D
12th well: N/A
13th well: small (100 bp) ladder
14th well: N/A
15th well: large ladder (10 kb)
16th well: N/A
The electrophoresis tank was then run at 80 V for 1 hour.
Quantified colonies A, B, C, and D of pET-Duet-1 plasmids assembled with ATPG and NRPS, and Clones A, B, C, and D of pRSF-Duet-1 plasmids assembled with DHQS using the NanoDrop Spectrophotometer:
PET-Duet-1 ATPG/NRPS Clone A: 171.1 ng/µL
260:280 ratio: 2.15 - protein (280 and also 220 – 10X bigger – peptide bond)
260:230 ratio: 2.05
PET-Duet-1 ATPG/NRPS Clone B: 92.4 ng/µL
260:280 ratio: 1.99
260:230 ratio: 1.41
PET-Duet-1 ATPG/NRPS Clone C: 121.8 ng/µL
260:280 ratio: 1.99
260:230 ratio: 0.97
PET-Duet-1 ATPG/NRPS Clone D: 128.0 ng/µL
260:280 ratio: 1.89
260:230 ratio: 1.61
PRSF-Duet-1 DHQS Clone A: 430.1 ng/µL
260:280 ratio: 1.91
260:230 ratio: 2.14
PRSF-Duet-1 DHQS Clone B: 335.4 ng/µL
260:280 ratio: 1.96
260:230 ratio: 2.04
PRSF-Duet-1 DHQS Clone C: 277.8 ng/µL
260:280 ratio: 1.88
260:230 ratio: 1.97
PRSF-Duet-1 DHQS Clone D: 215.9 ng/µL
260:280 ratio: 1.89
260:230 ratio: 1.90
Dyeing the 1% agarose gel:
Nucleic acid dye (15 µL) was added to 1X buffer TAE (150 mL). This was then poured over the 1% agarose gel, and rocked for 30 minutes.
Sent off pET-Duet-1 plasmids assembled with ATPG/NRPS (clones A and B), and the pRSF plasmids assembled with DHQS (clones A and B) for sequencing
PET-Duet-1 ATPG/NRPS clone C (9.85 µL, 121.8 ng/µL) was added to nuclease-free water (5.15 µL), giving a more diluted concentration of PET-Duet-1 ATPG/NRPS clone C (15 µL, 80 ng/µL). This was repeated once more. The two eppendorf tubes containing diluted PET-Duet-1 ATPG/NRPS clone C were sent to Eurofins Genomics GATC Services for Custom DNA sequencing. Each clone was sequenced forwards and backwards: one of the tubes had MCS1 (containing ATPG) sequenced – hence the forwards primer was petup and the reverse primer was DuetDOWN1. The other tube had MCS2 (containing NRPS) sequenced: the forwards primer was DuetUP2 and the reverse primer was T7term.
PET-Duet-1 ATPG/NRPS clone D (9.375 µL, 128.0 ng/µL) was added to nuclease-free water (5.625 µL), giving a more diluted concentration of PET-Duet-1 ATPG/NRPS clone D (15 µL, 80 ng/µL). This was repeated once more. The two eppendorf tubes containing diluted PET-Duet-1 ATPG/NRPS clone D were sent to Eurofins Genomics GATC Services for Custom DNA sequencing. Each clone was sequenced forwards and backwards: one of the tubes had MCS1 (containing ATPG) sequenced – hence the forwards primer was petup and the reverse primer was DuetDOWN1. The other tube had MCS2 (containing NRPS) sequenced: the forwards primer was DuetUP2 and the reverse primer was T7term.
PRSF-Duet-1 DHQS clone A (2.79 µL, 430.1 ng/µL) was added to nuclease-free water (12.21 µL), giving a more diluted concentration of PRSF-Duet-1 DHQS clone A (15 µL, 80 ng/µL). The eppendorf tube containing diluted PRSF-Duet-1 DHQS clone A was sent to Eurofins Genomics GATC Services for Custom DNA sequencing. The clone was sequenced forwards and backwards: this tube had MCS1 (containing DHQS) sequenced – hence the forwards primer was ACYCDuetUP1 and the reverse primer was DuetDOWN1.
PRSF-Duet-1 DHQS clone B (3.58 µL, 335.4 ng/µL) was added to nuclease-free water (11.42 µL), giving a more diluted concentration of PRSF-Duet-1 DHQS clone B (15 µL, 80 ng/µL). The eppendorf tube containing diluted PRSF-Duet-1 DHQS clone B was sent to Eurofins Genomics GATC Services for Custom DNA sequencing. The clone was sequenced forwards and backwards: this tube had MCS1 (containing DHQS) sequenced – hence the forwards primer was ACYCDuetUP1 and the reverse primer was DuetDOWN1.
Barcodes:
2HF443 - pET ATPG C FWD – petup
2HF444 – pET ATPG C REV – DuetDOWN1
2HF445 - pET ATPG D FWD - petup
2HF446 - pET ATPG D REV - DuetDOWN1
2HF447 - pET NRPS C FWD - DuetUP2
2HF448 – pET NRPS C REV - T7term
2HF449 - pET NRPS D FWD - DuetUP2
2HF450 - pET NRPS D REV - T7term
2HF451 - pRSF DHQS A FWD – ACYCDuetUP1
2HF452 - pRSF DHQS A REV - DuetDOWN1
2HF453 - pRSF DHQS B FWD - ACYCDuetUP1
2HF454 - pRSF DHQS B REV - DuetDOWN1
At home:
30/07/21 - Maia
Editing of Thanogen composite parts
The IDT ordering tool was used again to find the difficulties as mentioned above with the Thanogen composite parts. After these were highlighted, the troublesome parts were codon optimised and edited to consist of less repeats (which was the initial issue). After all of this editing, the ‘difficulty to manufacture’ score actually increased. After trial and error for some time with these parts, it was decided best to consult one of our supervisors next week for some help.
Saturday 31st July
KCL educational lecture
AJ designed and created 5 slides for the KCL Biologix educational lecture.
August
Sunday 1st August
KCL educational lecture
AJ wrote the script for each of her 5 slides of the KCL Biologix educational lecture. Continued work on other areas of the project.
Week 9:
Monday 2nd August
02/08/21 - Anna, Maia, Julia, AJ, Yoricka.
Sequencing results from Eurofins Genomics:
None of the bands were in the right place – some of the bands were the original plasmid – some of them were the linear plasmid – but neither NRPS or DHQS was inserted into their respective plasmids correctly.
Membrane wash solution was likely the cause
The gel extractions went wrong.
Making 1.5% agarose gels
Agarose (2.25 g) was added to 1X TAE buffer (150 mL). The resulting mixture was microwaved (swirled at 30 second intervals) until the agarose dissolved. The solution was left to cool, and then a portion of it was poured into a gel rig. This gel rig had mega wells (whereby there were 3 mega-wells made up of 4 normal-sized wells each, as well as there being two normal-sized wells on either side for the ladders). Once the solution had set in the rig, the agarose gel was placed in the fridge.
Add 1 microlitre then incubate for 20/30 minutes at 37 degrees.
DpnI treatment of the PCR. We set up a tube for pRSF
We added some of the pCR product, BSA, water, restriction enzyme buffer. We still have 15 microlitres of everything left. We will add 1 microlitre of new DpnI to this – the concentrations will be slightly off, but we will have 16 microlitres in total.
All the DNA will be fine. Diamond Dye should be fine. Doubt about enzymes (we can get more of these – stock freezer, or borrowing someone elses), doubt about Gibson Assembly.
Try doing Gibson assembly (there is no control for this). There is no way to test it – if it works, you’ll see colonies. If you see no colonies, it won’t have worked.
We can test some of the restriction enzymes.
Carry out digests with the restriction enzymes, run on a gel, see if the restriction digest has worked (this tells us whether the enzymes are still viable).
KCL educational lecture
AJ made recordings for each of her 5 slides in the KCL educational lecture.
Tuesday 3rd August
02/08/21 - Maia, Julia, Anna, AJ, Yoricka.
1.5 % agarose gel electrophoresis of O-MT, DHQS, and NRPS PCR products
1X TAE buffer was poured into the gel rig until it covered the 1.5 % agarose gel. At the same time, the Quick-load Purple 1 kb Plus DNA ladder (100bp- 10kb in size), and the dye (Blue/Orange 6X loading dye) were defrosted.
The three DHQS PCR replicates (50 µL each) were added to the Blue/Orange 6X loading dye (30 µL), and the resulting mixture was mixed by flicking. This mixture (80 µL) was then added to the first mega well in the agarose gel. This procedure was repeated for the NRPS and O-MT PCR products.
The already-dyed large (1,000 – 10 kb in size) DNA ladder (10 µL) was then added to the 1st and 16th wells in the agarose gel.
The PCR gene products and ladders were placed in the following order in the gel:
1st well: large ladder
2nd-5th wells: DHQS
6th well: blank
7th - 10th wells: NRPS
11th well: blank
12th-15th wells: O-MT
16th well: large ladder
The electrophoresis tank was left to run at 80V for 90 minutes. Half-way through the procedure, more TAE buffer was used to cover the gel, following the advice of our supervisor.
Dyeing the 1.5% Agarose Gel
Nucleic acid dye (15 µL) was then added to the resultant 1X buffer TAE (150 mL). This was then poured over the 1.5% agarose gel, and rocked for 30 minutes.
Gel Visualisation
Our supervisor helped to analyse the gel image. It was concluded that the separation of the DNA worked, however the stains were not very clear. Gel extraction was attempted.
Gel Extraction
The bands containing the O-MT, NRPQ, and DHQS genes were excised from the gel.
Gel Slice Preparation
Weighing the masses of each gel slice:
DHQS: 0.524 g
NRPS: 0.546 g
O-MT: 0.412 g
Membrane binding solution (524 µL, 546 µL, and 412 µL) was added to the DHQS, NRPS, and O-MT gel slices respectively, in a ratio of 1 µL of Membrane Binding Solution per 1 mg of gel slice. The resulting mixtures were vortexed, then incubated at 50 °C for 10 minutes, at which time the gel slices had dissolved.
To bind the DNA, the dissolved gel mixture was poured into a minicolumn assembly, then kept at room temperature for 1 minute. This was centrifuged at 16,000 x g for 1 minute, then the flowthrough was discarded. (Ethanolic) membrane Binding solution (55 µL) was then added to the DHQS product again, since some gel slice was remaining in the tube. This was incubated at 60 °C for 3 minutes until the gel had dissolved. The dissolved gel mixture was poured into a minicolumn assembly again, kept at room temperature for 1 minute, centrifuged at 16,000 x g for 1 minute, and the flowthrough discarded.
Next, (ethanolic) Membrane Wash Solution (700 µL) was added to each of the DHQS, NRPS, and O-MT products, and the minicolumn assembly centrifuged at 16,000 x g for 1 minute. The flowthrough was discarded, then (ethanolic) Membrane Wash Solution (500 µL) was added to each of the DHQS, NRPS, and O-MT products again, and the minicolumn assembly centrifuged at 16,000 x g for 5 minutes. The flowthrough was discarded, and the column assembly was centrifuged at 16,000 x g for 1 minute.
The minicolumn was then transferred to a 1.5 microcentrifuge tube. Nuclease-free water (50 µL) was added, the tube incubateed at room temperature for 1 minute, then centrifuged at 16,000 x g for 1 minute. The DNA was then quantified using a NanoDrop Spectrophotometer.
Quantifying DHQS, NRPS, and O-MT using the NanoDrop spectrophotometer
DHQS: 36.3 ng/µL
260/280 ratio: 1.70 (compared to ideal of 1.8)
260/230 ratio: 1.45
This has the weakest concentration and 260/280 ratio.
NRPS: 26.4 ng/µL
260/280 ratio: 1.76 (compared to ideal of 1.8)
260/230 ratio: 1.36
O-MT: 23.0 ng/µL
260/280 ratio: 1.88 (compared to ideal of 1.8)
260/230 ratio: 1.17
Making a 1 % agarose gel
Agarose (1.5 g) was added to 1X TAE buffer (150 mL). The resulting mixture was microwaved (being swirled at 30 second intervals) until the agarose dissolved. The solution was left to cool, and then it was poured into the gel rig. The gel rig was taped to make larger wells. Once the solution had set, the agarose gel was placed in the fridge.
Gibson Assembly of NRPS into ATPG-containing pET-Duet-1
NRPS (2.22 µL), pET-Duet-1 ATPG (5 µL), and nuclease-free water (2.78 µL) was added to the MasterMix (10 µL). The resulting mixture was incubated at 50 °C for 30 mins, then cooled on ice. 18 µL of the product was stored at 4 °C for future use. 2 µL was used for transformation, as outlined below.
Gibson Assembly of DHQS into pRSF-Duet
DHQS (1.377 µL), pRSF-Duet (5 µL), and nuclease-free water (3.623 µL) was added to MasterMix (10 µL). The resulting mixture was incubated at 50 °C for 30 mins, then cooled on ice. 18 µL of the product (20 µL) was stored at 4 °C for future use. 2 µL was used for transformation, as outlined below.
Transformation of pET-Duet-1 plasmid assembled with ATPG and NRPS:
The chemically competent DH5α cells (50 µL) were thawed on ice for 5 minutes. The assembled pET-Duet-1 plasmids (2 µL) were then added to these cells, and the tube containing the resulting mixture was flicked gently 5 times. The mixture was then placed on ice for 30 minutes. Following this, the cells were heatshocked at 42 °C for 35 seconds, before being placed on ice for an additional 2 minutes. Next, SOC media (950 µL) was added to the tubes, and the tubes were taped horizontally (to improve aeration, since increased movement of liquid results in more mixing) in a 37 °C incubator shaking at 130 rpm for 1 hour. Following this, 100 microlitres of the cells were spread onto plates with ampicillin. This was repeated for another plate, then the plates were then incubated overnight at 37 °C.
Transformation of pRSF-Duet-1 plasmid assembled with DHQS:
The chemically competent DH5α cells (50 µL) were thawed on ice for 5 minutes. The assembled pRSF-Duet-1 plasmids (2 µL) were then added to these cells, and the tube containing the resulting mixture was flicked gently 5 times. The mixture were then placed on ice for 30 minutes. Following this, the cells were heatshocked at 42 °C for 35 seconds, before being placed on ice for an additional 2 minutes. Next, SOC media (950 µL) was added to the tubes, and the tubes were taped horizontally (to improve aeration, since increased movement of liquid results in more mixing) in a 37 °C incubator shaking at 130 rpm for 1 hour. Following this, 100 microlitres of the cells were spread onto a plate with kanamycin. This was repeated for another plate, then the plates were then incubated overnight at 37 °C.
IGEM Stuttgart meeting today for a potential collaboration
Wednesday 4th August
Testing restriction enzyme function using control plasmids
The Restriction Enzyme 10X Buffer E (2 µL), nuclease-free water (15 µL), and finally HindIII Restriction enzyme (1 µL, 10 u/µL) was added to a control plasmid (puc19) (2 µL). The resulting mixture was placed in the incubator at 37 °C for 2 hours.
This procedure was repeated for the NdeI Restriction enzyme, using restriction Enzyme 10X Buffer D4 (2 µL).
Also we will need supervisors on Monday and Tuesday.
We had a few colonies (we are not doing gibson again).
Make more plates again tonight (overnights) to see if we can get more colonies (redo the transformation).
Today – we did overnights and transformation (same as yesterday).
Delivered the KCL Biologix Lecture (2 pm, zoom).
Thursday 5th August
Miniprep the assembled plasmids
The DH5α bacterial colonies grown overnight were taken out of the incubator and centrifuged at 15,000 x g for 10 minutes. All pET-Duet-1 colonies (A, B, C, and D) were transparent following centrifugation. The single pRSF colony was also transparent following centrifugation. The LB broth was then discarded from each tube (the falcon tubes were held upside town on a paper towel to dry the pellets), PBS (500 µL) was added to each pellet, and then the resulting mixture was vortexed until each pellet was resuspended. Each mixture was microcentrifuged again at maximum speed for 15 seconds. At this point, the PBS should have been discarded, however we forgot to do this for pET-Duet-1 clones A, B, and C (the PBS was discarded for pET-Duet-1 clone D, and the pRSF-Duet-1 clone). The procedure was carried on as normal. Cell lysis buffer (200 µL) was added to each of the pellets. The resulting solutions were mixed by inverting 6 times, flicked, and then mixed by pipetting until the pellets were resuspended. Neutralisation solution (350 µL) was added to each mixture, and then the resulting mixtures were also inverted and flicked until they turned completely yellow. These mixtures were then microcentrifuged at maximum speed for 3 minutes. The supernatant was pipetted into minicolumns and then each minicolumn was microcentrifuged at maximum speed for 15 seconds. The flowthough was discarded, the Endotoxin Removal Wash (200 µL) was added to each minicolumn, and then the minicolumns were centrifuged at maximum speed for 15 seconds again. Column Wash Solution (400 µL) was then added to each minicolumn, before centrifuging at maximum speed for 30 seconds. The minicolumns were then transferred to a clean eppendorf tube, then Elution Buffer (30 µL) was added directly to each minicolumn matrix. These matrices were left to stand at room temperature for 1 minute. To elute the plasmid DNA, the eppendorf tubes were microcentrifuged at maximum speed for 15 seconds. Any miniprep not used in the restriction digest was placed in the freezer.
Quantified colonies A, B, C, and D of pET-Duet-1 plasmids assembled with ATPG and NRPS, and the single clone of pRSF-Duet-1 plasmid assembled with DHQS using the NanoDrop Spectrophotometer:
PET-Duet-1 ATPG/NRPS Clone A: 35.2 ng/µL
260:280 ratio: 1.93 - protein (280 and also 220 – 10X bigger – peptide bond)
260:230 ratio: 1.12
PET-Duet-1 ATPG/NRPS Clone B: 25.9 ng/µL
260:280 ratio: 1.99
260:230 ratio: 1.41
PET-Duet-1 ATPG/NRPS Clone C: 46.2 ng/µL
260:280 ratio: 1.73
260:230 ratio: 0.76
PET-Duet-1 ATPG/NRPS Clone D: 186.7 ng/µL
260:280 ratio: 1.80
260:230 ratio: 1.40
PRSF-Duet-1 DHQS Clone A: 261.2 ng/µL
260:280 ratio: 1.93
260:230 ratio: 1.99
Restriction digest - Assembled pET-Duet-1 plasmids:
The Restriction Enzyme 10X Buffer D5 (2 µL), nuclease-free water (15 µL), and finally the MluI Restriction Enzyme (1 µL, 10 µ/µL) was added to the miniprep product (2 µL). This was repeated for each of the assembled pET-Duet-1 miniprep products. The four resulting mixtures were placed in the incubator at 37 °C for 2 hours.
Restriction digest - Assembled pRSF-Duet-1 plasmids:
The Restriction Enzyme 10X Buffer E6 (2 µL), nuclease-free water (15 µL), and finally the HindIII Restriction Enzyme (1 µL, 10 µ/µL) was added to the miniprep product (2 µL). This was repeated for each of the assembled pRSF-Duet-1 miniprep products. The four resulting mixtures were placed in the incubator at 37 °C for 2 hours.
Running the gel
Loading dye (4µL) was added to each of the digests (20 µL). Loading dye (1 µL) was also added to the small 100 bp ladder (5 µL). The large ladder (5 µL) was already pre-dyed.
Then the dyed restriction digests (15 µL), the dyed small 100 bp ladder (6 µL), and the large 10 kb ladder (5 µL) were added to the 1% agarose gel (submerged in 1X TAE buffer) in the following order:
1st well: large (10 kb) ladder
2nd well: N/A
3rd well: pET-Duet-1 ATPG/NRPS plasmid digest product, colony A
4th well: N/A
5th well: pET-Duet-1 ATPG/NRPS plasmid digest product, colony B
6th well: N/A
7th well: N/A
8th well: pET-Duet-1 ATPG/NRPS plasmid digest product, colony C
9th well: N/A
10th well: pET-Duet-1 ATPG/NRPS plasmid digest product, colony D
11th well: N/A
12th well: pRSF-Duet-1 DHQS plasmid digest product, colony A
13th well: N/A
14th well: large ladder (10 kb)
15th well: N/A
16th well: N/A
The electrophoresis tank was then run at 80 V for 1 hour.
Dyeing the 1% agarose gel:
Nucleic acid dye (35 µL - since the bands were previously not visible using 15 µL of dye) was added to 1X buffer TAE (150 mL). This was then poured over the 1% agarose gel, and rocked for 30 minutes.
After 30 minutes of dying.
After an additional 15 ul of dye and 15 minutes on the shaker. Gel image quality remained at about the same level.
Gels were very difficult to visualise. We will rely on sequencing to determine whether gibson assembly worked successfully.
Sent off pET-Duet-1 plasmid assembled with ATPG/NRPS (clones D), and the pRSF plasmid assembled with DHQS (clones A) for sequencing
PET-Duet-1 ATPG/NRPS clone D (6.4 µL, 186.7ng/µL) was added to nuclease-free water (8.6 µL), giving a more diluted concentration of PET-Duet-1 ATPG/NRPS clone C (15 µL, 80 ng/µL). The eppendorf tube containing diluted PET-Duet-1 ATPG/NRPS clone D was sent to Eurofins Genomics GATC Services for Custom DNA sequencing. Clone A was sequenced forwards and backwards: one of the tubes had MCS1 (containing ATPG) sequenced – hence the forwards primer was petup and the reverse primer was DuetDOWN1. The other tube had MCS2 (containing NRPS) sequenced: the forwards primer was DuetUP2 and the reverse primer was T7term.
PRSF-Duet-1 DHQS clone A (4.6 µL, 261.2 ng/µL) was added to nuclease-free water (10.4 µL), giving a more diluted concentration of PRSF-Duet-1 DHQS clone A (15 µL, 80 ng/µL). The eppendorf tube containing diluted PRSF-Duet-1 DHQS clone A was sent to Eurofins Genomics GATC Services for Custom DNA sequencing. The clone was sequenced forwards and backwards: this tube had MCS1 (containing DHQS) sequenced – hence the forwards primer was ACYCDuetUP1 and the reverse primer was DuetDOWN1.
Barcodes:
2HF468 - pET ATPG Clone D FWD – petup
2HF469 – pET ATPG Clone D REV – DuetDOWN1
2HF470 - pET NRPS Clone D FWD - DuetUP2
2HF471 - pET NRPS Clone D REV - T7term
2HF472 – pRSF DHQS Clone A FWD - ACYCDuetUP1
2HF473 – pRSF DHQS Clone A REV – DuetDOWN1
Friday 6th August
UNSW meeting: potential collaboration
Not allowed in the lab (no lab supervision) - today was an ‘admin’ day.
Saturday 7th August
Did more work on the project.
Sunday 8th August
More work on the project!
Week 10:
Monday 9th August
09/08/21 - Anna, Maia, Julia, Yoricka, AJ
Transformations of Plasmids into BL21 cells
Competent BL21 cells were sourced, moved from –80 °C to –20 °C over lunch, before being thawed on ice.
3 kanamycin agar plates (and 5 ampicillin agar plates were placed in an incubator at 37 °C.
The following miniprepped plasmids were then used in the transformation procedure:
pET_ATPG (302.8 ng/µL)
pET_ATPG_NRPS (186.7 ng/µL)
pRSF_DHQS (261.2 ng/µL)
Two separate dilutions were carried out for the transformation of each plasmid into competent BL21 cells.
For the transformation of the pET_ATPG plasmid into BL21 competent cells:
To carry out a 1 in 300 dilution, the miniprepped plasmid (1 µL) was added to nuclease-free water (299 µL). Then 1 ng of the diluted plasmids (1 µL) were added to BL21 competent cells (50 µL).
For the 1 in 50 dilution, the miniprepped plasmid (1 µL) was added to nuclease-free water (49 µL). Then 10 ng of the diluted plasmids (1.66 µL) was added to BL21 competent cells (50 µL).
For the transformation of the pET_ATPG_NRPS plasmid into BL21 competent cells:
To carry out a 1 in 200 dilution, the miniprepped plasmid (1 µL) was added to nuclease-free water (199 µL). Then 1 ng of the diluted plasmids (1.07 µL) were added to BL21 competent cells (50 µL).
For the 1 in 50 dilution, the miniprepped plasmid (1 µL) was added to nuclease-free water (49 µL). Then 10 ng of the diluted plasmids (2.68 µL) were added to BL21 competent cells (50 µL).
For the transformation of the pRSF_DHQS plasmid into BL21 competent cells:
To carry out a 1 in 300 dilution, the miniprepped plasmid (1 µL) was added to nuclease-free water (299 µL). Then 1 ng of the diluted plasmids (1.15 µL) were added to BL21 competent cells (50 µL).
For the 1 in 50 dilution, the miniprepped plasmid (1 µL) was added to nuclease-free water (49 µL). Then 10 ng of the diluted plasmids (1.91 µL) was added to BL21 competent cells (50 µL).
All the resulting plasmid/BL21 cell mixtures were then placed on ice for 20-30 minutes, heat shocked at 42 °C for 45 seconds, placed back on ice for 2 minutes. LB broth (600 µL) was added to each of the 6 tubes containing the transformed BL21 cells. Each of the 6 tubes (two separate dilutions for each plasmid) were then incubated (taped horizontally, to improve aeration) at 37 °C at 130 rpm for 45 minutes.
Following this, the cells transformed with pRSF_DHQS (50 µL) were spread onto kanamycin plates. The cells transformed with pET plasmids (50 µL) were spread onto ampicillin plates. A stock containing nuclease-free water (50 µL) and LB broth (600 µL) were also created, then this stock (150 µL) was added to one ampicillin and one kanamycin plate each. All of the plates were then incubated overnight at 37 °C.
Tuesday 10th August
10/08/21 - Anna, Maia, Julia, Yoricka, AJ
No colonies were present on any of the plates that were incubated overnight. We decided to streak new plates with more transformed BL21 cells.
For each of the 3 plasmids (pET_ATPG, pET_ATPG_NRPS, and pRSF_DHQS), three agar plates of transformed BL21 cells were set up.
Plate 1: 3x more DNA on the 10 ng DNA plate compared to yesterday. BL21 cells (150 µL) transformed with plasmid (10 ng) in LB broth were spread onto agar plates.
Plate 2: 8x more DNA on the 10 ng DNA plate compared to yesterday. BL21 cells (400 µL) transformed with plasmid (10 ng) in LB broth were spread onto agar plates.
Plate 3: 11 x more DNA on the 1 ng DNA plate compared to yesterday. BL21 cells (550 µL) transformed with plasmid (1 ng) in LB broth were spread onto agar plates.
Two controls were also set up: the nuclease-free water/LB broth stock (200 µL) made yesterday was added to one ampicillin agar plate, and one kanamycin agar plate.
A final control was also set up: LB broth (150 µL) was added to BL21 competent cells (50 µL), and the resulting solution was plated onto agar plates which contained NO antibiotics.
All of the resulting plates were incubated overnight at 37 °C.
Wednesday 11th August
11/08/21 - Anna, Maia, Julia, Yoricka, AJ
In the morning, the plates with transformed BL21 cells spread on them the night before had no colonies present. Only one plate had colonies growing on it: this was the plate which contained NO antibiotics, whereby the BL21 cells added to this plate had not been transformed with any plasmids.
Sequencing results
Sequencing results came back from Eurofins Genomics GATC Services. According to the results, neither the assembly of DHQS into pRSF-Duet-1 nor the assembly of NRPS into pET-Duet-1_ATPG was successful.
Transformation of pET-Duet-1_ATPG Plasmids into BL21 competent cells
Competent BL21 cells were thawed on ice for 30 minutes.
The following plasmids were transformed into competent BL21 cells:
pET-Duet-1_ATPG (302.8 ng/µL)
pET-Duet-1
The pET-Duet-1_ATPG plasmid (2 µL, 302.8 ng/µL) were added to BL21 competent cells (50 µL). The pET-Duet-1 plasmids (2 µL, 268 ng/µL) was also added to BL21 competent cells (50 µL).
The resulting plasmid/BL21 cell mixtures were then placed on ice for 20-30 minutes, heat shocked at 42 °C for 45 seconds, then placed back on ice for 2 minutes. LB broth (200 µL) was added to each of the two tubes containing the transformed BL21 cells. The two tubes were then incubated (taped horizontally, to improve aeration) at 37 °C at 130 rpm for 45 minutes.
Following this, the cells transformed with pET plasmids in LB broth (200 µL) were spread onto two ampicillin plates each. Then an agar plate was made: agar was heated to 60 °C until it had dissolved. Then, after having sterilised all the equipment, the molten agar was poured into three plates. Competent BL21 cells (50 µL) were added to LB broth (200 µL). This mixture (200 µL) was then added to one of these agar plates which did not select for resistance to antibiotics. All three of the plates were then incubated overnight at 37 °C.
Thursday 12th August
12/08/21 - Maia, Julia, Yoricka, AJ
This morning, none of the plates spread with transformed BL21 cells had any colonies growing on them. It was thus concluded that the BL21 cells we had been using were viable, rather than competent. To overcome this obstacle, we sourced a different batch of competent BL21 cells and repeated the transformation. Because one of the researchers in our biology department was using these competent BL21 cells successfully at the same time as us, she gave us a slightly different protocol to use to ensure that our methods were optimal.
Transformation of pET-Duet-1_ATPG Plasmids into BL21 competent cells
Competent BL21 cells were thawed on ice for 30 minutes.
The following plasmids were transformed into competent BL21 cells:
pET-Duet-1_ATPG (302.8 ng/µL)
pET-Duet-1
The pET-Duet-1_ATPG plasmid (1 µL, 302.8 ng/µL) were added to BL21 competent cells (50 µL). The pET-Duet-1 plasmids (1 µL, 268 ng/µL) was also added to BL21 competent cells (50 µL).
The resulting plasmid/BL21 cell mixtures were then placed on ice for 30 minutes, heat shocked at 42 °C for 30 seconds, then placed back on ice for 5 minutes. LB broth (500 µL) was added to each of the two tubes containing the transformed BL21 cells. The two tubes were then incubated (taped horizontally, to improve aeration) at 37 °C at 130 rpm for 1 hour.
Following this, the cells transformed with pET plasmids in LB broth (100 µL) were spread onto two ampicillin plates each. Competent BL21 cells (50 µL) were then added straight to LB broth (500 µL). This mixture (100 µL) was added one of these agar plates which did not select for resistance to antibiotics. All three of the plates were then incubated overnight at 37 °C.
Friday 13th August
No visible colonies on any of our transformed plates. So our only successfully cloned in gene was ATPG. We were of course bummed that this was our final result, but were happy to at least have cloned in one gene successfully into one of our plasmids!
Saturday 14th August
Continued project work.
Sunday 15th August
More project work, more focused now on human practices, education, and outreach now that the wetlab portion of our project had gone through its 10 weeks.