Notebook

Abstract

Here we describe our weekly notebook regarding what we’ve done in the lab, from the beginning of the project and all the way till Wikifreeze week!

Introduction

This is our notebooks for the entire project.

Week 23 – June 7th to June 13th
Learned how to streak bacteria from freeze stock onto a LA plate and how to make ON cultures from the bacteria grown on the LA plate. From this ON culture we made our first freeze stock of 250µL 50% glycerol and 750µL ON culture.
This was our first freeze stock, named freeze stock 1.
This week a ON culture of an AMP resistant Top10 strain from SDU-iGEM2019’s part 11 was prepared, which was miniprepped to make Part 1 (Bba_J61002 with Bba_J23102) and made a freeze stock from freeze stock 2.
To learn how to preform gel electrophoresis, part 1 (Bba_J61002 with Bba_J23102) was loaded onto a 1% agarose gel.

Week 24 – June 14th to June 20th
It was taught how to use restriction enzymes to cut a plasmid, which in the future will be used for transformation.
After cutting the plasmid it was loaded on a gel and purified afterwards. Solutions of 0.1M MgCl₂ and 0.1M CaCl₂ was prepared for use in CaCl₂ competent cells.
Also learned how to make our own LA-plates with selection, here using kanamycin.

Week 25 – June 21st to June 27th
To further prepare for transformation, it was taught how to make TSB buffer. ON culture of SDU-iGEM 2019’s part 10 was miniprepped to create part 2 (Bba_J61002 with Bba_J23106).
From the iGEM distribution plate 3 2019 well 2M, which contained the LacI gene (Bba_I732820), part 3 (Bba_I732820 in pSB1C3) was taken. This week the first transformation was made, here part 3 (Bba_I732820 in pSB1C3) was transformed into a Top10 culture. After transformation the bacteria was plated on LB plates with ampicillin, however, no colonies formed due to using the wrong antibiotics.
We cleaved the part 2 (Bba_J61002 with Bba_J23106) we had created this week. First, we cleaved using SpeI and XbaI, which was not correct, hereafter it was redone correctly.

Week 26 – June 28th to July 4th
This week we transformed part 3 into a Top10 culture again and plated the bacteria out on chloramphenicol plates. A freeze stock was made of the Top10 containing part 3 (Bba_I732820 in pSB1C3).
Using Taq PCR and electrophoresis we also confirmed our primers oligo 3 (suffix primer) and oligo 4 (prefix primer) are correct by testing them on our part 2 (Bba_J61002 with Bba_J23106).
We learned how to ligate two parts together, here with part 2 and part 3 (Bba_I732820 in pSB1C3) to a new part called part 4 (Bba_J61002 with Bba_I732820). The ligation product was transformed into Top10 culture.

Group DKM:
This week we also received our first biobricks PsiDK and PsiM, they were amplified and purified so they were ready for use (BBa_K4059010).

Week 27 – July 5th to July 11th
Group PsiDKM:
This week, using Phusion PCR mix, we put together our biobricks PsiDK and PsiM to create PsiDKM (BBa_K4059010). We cleaved our part 4 (Bba_J61002 with Bba_I732820) (vector) and PsiDKM (insert) and ligated them together. The ligation product was transformed into Top10 and named part 5 (BBa_K4059037). The bacteria was plated and haven harvested for use in colony PCR. Correct transformed bacteria from the colony PCR was made into a ON culture. The cultures were induced with IPTG as it should induce PsiDKM. To test the induction, we peformed SDS-page.
The SDS-page results were unfortunately not conclusive as the expected bands were present both in IPTG induced samples and non-induced samples.
Group PsiH: We transformed the future backbone for our chaperone proteins into Top10 (SOP14). The backbone was pSB1C3 containing biobrick BBa_I0500, there BBa_I0500 consists of a pBad/araC promoter system for E. coli. The plasmid was obtained from the iGEM 2019 plate 5 well 18M. From the successful transformation of pSB1C3+BBa_I0500, an overnight culture was grown (SOP4) and used for miniprep (SOP7) and freeze stock preparation (SOP5) for future use. Furthermore, we prepared a freeze stock of E. coli strain ER2566, our future protein expression strain.

Week 28 – July 12th to July 18th
Group PsiDKM:
Gel electrophoresis showed part 5 (BBa_K4059010) did not have the correct length, therefore we redid the ligation to recreate part 4 (Bba_J61002 with Bba_I732820) and ligated it together with our PsiDKM insert again. The ligation product was transformed into Top10 again.
Group PsiH:
pSB1C3 backbone with BBa_I0500 was cut with the FastDigest enzymes SpeI and PstI (SOP12) to prepare for insertion of GroES/EL biobrick. The gel-electrophoresis (SOP 8) showed a band just above 3000 bp (expected length 3270 bp), which was purified.
Furthermore, the backbone J61002 which contains medium cons. promoter (J23106) and LacI (I732820) was miniprepped from ON-culture (SOP 7) and cut with SpeI and PstI (SOP 12). The gel-electrophoresis (SOP 8) showed bands at just above 3000 bp (expected length 3310bp), which was purified (SOP 6). The purpose was to prepare a backbone for PsiH and CPR, before their arrival.

Week 29 – July 19th to July 25th
Group PsiDKM To test if insert and vector length was correct after the ligation and transformation, we cleaved part 5 with EcoRI + PstI, EcoRI + XbaI, SpeI + PstI, PstI and EcoRI. Gel electrophoresis did show bands were not the correct length.
Group PsiH: We received the biobricks for PsiH, CPR and GroES/EL. We resuspended the biobricks to receive a stock concentration of 50 ng/μL and further diluted the biobricks to working concentrations of 0,5 ng/μL. We also received overhang oligo sets for PsiH N-terminal 2-7 and DK N-terminal 2-7, which were diluted to working concentrations of 10 µM. We amplified GroES/EL by Phusion PCR (SOP 11). The PCR product was run on an agarose gel, purified, and cut with FastDigest enzymes XbaI and PstI. The cut products were also run on an agarose gel and purified (SOP12, SOP8 and SOP6). Afterward, GroEL/ES was ligated into pSB1C3 + BBa_I0500 (SOP 13), and the resulting plasmid was transformed into E. coli Top10 (SOP 14) (BBa_4059062). Single colonies from transformation with GroEL/ES were streaked out on agar plates and colony-PCR was performed the following day (SOP 9).
To obtain the T7-promoter + RBS (BBa_K4059009) from the DK biobrick, we performed PCR with different overhang oligos (N-terminal 2-7) on the DK-biobrick. The gel showed PCR-products of expected length ~160 bp, which were cut from gel and purified (SOP 10, step 8.1-8.5). We now had a T7-promoter + RBS with overhangs complementary to N-term. 2-7 (named T7-overhang 2-7).
For PsiH, we performed PCR with different overhang oligos (N-terminals 2-7), which initially did not show any bands. We then performed gradient-PCR (different annealing temperatures) on PsiH with oligo N-terminal 2, which also showed no results, which probably was due to hairpin formation in the long single stranded overhang oligos. We overcame this problem by adding DMSO (Dimethyl-sulfoxide). The results for PsiH oligo N-term 2 showed that 7% DMSO and 60°C annealing temperature was appropriate. These conditions were then used to amplify PsiH with the remaining oligos (N-term. 3-7). This worked for N-terminal 3-7. We now had PsiH with overhangs containing N-terminal 2-7 (named PsiH-overhang 2-7).
We assembled T7-overhang 2 with PsiH-overhang 2 by overhang gradient PCR (SOP10, step 8.6-8.8). The best result was observed for annealing temperature at 64℃, where a clear band was seen at ～1800bp’s . The PCR-product was purified (SOP 6) and another PCR with our forward and reverse primer (annealing before iGEM standard prefix and after standard suffix, respectively). This showed amplification, which confirmed that our T7- and PsiH-overhang 2 had successfully been assembled. We then repeated the above T7- and PsiH-overhangs for 3-7. This gave us T7+RBS+PsiH with N-terminals 2-7 (BBa_numbers below). All of the biobricks were cut using FastDigest enzymes XbaI and PstI (SOP12) to prepare for ligation with backbone J61002 containing med. cons. promoter + lacI (J23106 + I732820). The T7+RBS+PsiH N-term 2 was ligated with this backbone (SOP13) and transformed into E. Coli Top10 (SOP14). Single colonies from transformation were streak plated and colony-PCR was performed the following day (SOP9) and showed no bands in the expected length of ～3100bp’s (LacI + PsiH). We decided to redo the backbone with LacI (J61002 + J23106 + I732820) and also the assembling of T7- and PsiH-overhang 2 by overhang PCR next week.

Week 30 – July 26th to August 1st
Group PsiDKM
Transformation was performed of Top10 cells.
Group PsiH
In order to remake the backbone for PsiH and CPR (J61002+J23106+I732820), we cut J61002+J23106 with FastDigest enzymes SpeI + PstI and pSB1C3 + I732820 (SOP12). The cut products were run on gel (SOP 8) and showed bands at expected lengths of ～2100 bp’s (for J61002+J23106) and ～1300 bp’s (for I732820). The cut products were purified (SOP6), ligated (SOP13) and transformed into E. coli Top10 (SOP14). Single colonies were streak plated and colony-PCR was performed (SOP9), showing correct lengths at around 1500bp’s for at least four colonies, D, F, G and H. ON-cultures were grown from these four colonies.
We transformed our GroEL/ES plasmid (BBa_K4059062) into E. Coli ER2566 to prepare for gene expression assays. From the overnight culture of this transformation, we prepared a freeze stock (SOP5) for later use. Using the same overnight culture, we performed SDS-page (SOP21). The result showed an over-expression of proteins around 57 kDa and 10kDa in the Arabinose-induced cultures compared to the non-induced culture and the Wild type ER2566. These protein sizes are consistent with our GroEL (57.3 kDa) and GroES (10.4 kDa), respectively.

Week 31 – August 2nd to August 8th
Group PsiDKM
To figure out what was wrong, our PsiDKM biobrick was amplifided using Taq RED mastermix PCR mix and send in for sequencing using primer sets seq_1, seq_2, seq_7 and seq_8.
Meanwhile, two new PsiDKM was made using Taq RED mastermix PCR and Phusion PCR, using Taq RED mastermix gave no results and using phusion the bands were too short when run on a gel.
Group PsiH:
Oligos for N-terminal modification of CPR (CPR overhang oligo N-terminal 2-5) and further N-terminal modifications of PsiH (PsiH overhang oligo N-terminal 7 (with different annealing sequence) and Deletion) were received and diluted to 10µM.
For PsiH, a new overhang PCR was run to assemble T7- and PsiH-overhang 2 again (SOP10). This showed good results, with bands at ～1800bp’s i gelelectrophoresis. The PCR-product was cut out and purified (SOP6). Then, the product, T7+RBS+PsiH-Nterm 2 (BBa_K4059039), was cut with FastDigest restriction enzymes XbaI and PstI to prepare for ligation into the backbone (SOP12).
With the new overhang oligos for PsiH (Nterm 7 and Nterm-Deletion), the first PCR on the IDT PsiH-biobrick was performed (SOP10, step 8.1-8.5). PsiH amplified with overhang oligo N-term-Deletion showed results at annealing temperatures of 60℃, and the band was cut and purified to use for secondary PCR reaction (SOP10, step 8.6-8.8) later.
Initially no bands were observed for PsiH amplified with overhang oligo N-term-7, probably due to too high annealing temperature (since the annealing sequence was redesigned to be shorter in order to prevent hairpin-formation). PCR-reactions were repeated as gradient-PCRs, which showed convincing bands at 54°C and 55°C. These bands were cut and purified and used right away for the secondary PCR reaction (SOP10, step 8.6-8.8) to assemble T7- and PsiH-overhang 7. The following gel-electrophoresis showed bands at ～1800bp’s as expected, and these were cut out and purified (SOP8 and SOP6). Now, T7+RBS+PsiH-Nterm 7 (BBa_K4059043) were assembled, but unfortunately, the concentrations were too low for restriction cutting.
For CPR, the initial overhang PCR’s (SOP10, step 8.1-8.5) with overhang oligos Nterm-2-5 showed no results, probably also due to too high annealing temperature. The same PCR was repeated as gradient-PCR, which resulted in bands of correct length at ～2300bp’s for CPR-overhang 2 and 5 at lower annealing temperatures, which were cut and purified (SOP10 and SOP6). The secondary overhang PCR to assemble T7- + CPR-overhang 2 and T7- + CPR-overhang 5, showed bands at ～2500bp’s as expected, and these were cut and purified. Thereby, we had assembled T7+RBS+CPR Nterm 2 (BBa_K4059048) and T7+RBS+CPR 5 (BBa_K4059051). Unfortunately, the concentrations obtained from gel-purification were too low to cut, so PCR will be repeated.
The PCR on CPR with overhang oligo N-term 3 and 4 (SOP10, step 8.1-8.5) was repeated several times with different annealing temperatures and DMSO-concentrations but no good results. We decided to discontinue the use of overhang oligos N-term 3 and 4 for CPR.

Week 32 – August 9th to August 15th
Group PsiDKM
A new attempted at making PsiDKM was made, here the bands was the correct length, but they were smeared. The same precedor was done, this time using phusion mastermix. Here bands were of the correct size and no smears were visible.
Both part 4 and the new PsiDKM was cleaved. Part 4 with SpeI and PstI, PsiDKM with XbaI and PstI. The cleaved products were run on a gel and then purified, as the bands are the correct size. Part 4 and PsiDKM was used for ligation and then transformed into Top10. The transformation was plated and then streak plated the next day.
Group PsiH:
The new backbone for PsiH and CPR (J61002 + J23106 + I732820) was miniprepped from ON-culture to obtain enough for cutting (SOP7). It was then cut with FastDigest enzymes SpeI and PstI, run on gel and purified (SOP12). The backbone was ligated with different previously prepared inserts (SOP13), T7+RBS+PsiH-Nterm 2 (BBa_K4059039) (both old, from week 29 and new version from week 31) and T7+RBS+PsiH-Nterm 3 (BBa_K4059040) (from week 29). The plasmids were then transformed into E. coli Top10 (SOP14). Streak plating of single colonies, and then colony-PCR the next day (SOP9), which showed bands at the expected lengths of ～3100bp’s (LacI + PsiH). From ON-cultures (SOP4) of the successfully transformed colonies, freeze-stocks were prepared, and plasmids were miniprepped and sent to sequencing (SOP7 and SOP17).
For PsiH, overhang PCR to assemble T7- and PsiH-overhang 7 was repeated to obtain more product (T7+RBS+PsiH-Nterm 7) (SOP10).
The same was done for T7- and CPR-overhang 2 and T7- and CPR-overhang 5. None of the mentioned overhang PCR reactions showed any correct bands on the following gel-electrophoresis, and a lot of smear was seen on gel-pictures. For this reason, we decided to repeat all of the primary PCRs (SOP10, step 8.1-8.5) using overhang oligos N-term 2-7+Deletion for PsiH, overhang oligos N-term 2 + 5 for CPR, and overhang oligos N-term 2-7+Deletion. For DK (to obtain T7-polymerase and RBS from this biobrick), the PCR products were run on agarose gels (SOP8), which were much longer than the regular gels, and also had different concentrations of agarose (0,7% for CPR and PsiH and 2% for T7). This way, we hoped to better separate the PCR products from any unspecific bands this way. The following gel-electrophoresis showed bands of correct length for PsiH-overhang 3, 5 and Deletion (～1800bp’s) and for T7-overhang 2-7 + Deletion (～160bp’s). The bands were cut out and purified (SOP6). For CPR with overhang oligos N-term 2 and 5, no bands were observed on agarose gel.

Week 33 – August 16th to August 22nd
Group PsiDKM
We did a colony PCR over the transformation of Part 5, part 5 was afterwards miniprepped and transformed into ER2566. Part 5 in ER2566 was induced with different concentrations of IPTG and samples was taken during different time stamps. These were run on an SDS-page gel but PsiD, PsiK, and PsiM are all similar in size and hard to tell apart. Therefore, qPCR preparation will begin next week.
Group PsiH:
Following the same procedure as last week, the backbone for PsiH and CPR (J61002 + J23106 + I732820) was miniprepped from ON-culture (SOP7), and cut with FastDigest enzymes SpeI and PstI, run on gel and purified (SOP6). The backbone was ligated with different previously prepared inserts, this time T7+RBS+PsiH-Nterm 4 (BBa_K4059036) (from week 29) and T7+RBS+PsiH-Nterm 5 (BBa_K4059042) (from week 29) (SOP13). The plasmids were then transformed into E. coli Top10 (SOP14). Streak plating of single colonies, and then colony-PCR the next day (SOP9), which showed bands at the expected lengths of ～3100bp’s (LacI + PsiH) for the T7+RBS+PsiH-Nterm 4 insert. For the Nterm 5 insert, the colony PCR was inconclusive.
The miniprepped plasmids from last week’s transformations, T7+RBS+PsiH-Nterm 2 (BBa_K4059039) (both old and new version) and T7+RBS+PsiH-Nterm 3 (BBa_K4059040) were cut with FastDigest enzymes X and P to verify the insert (SOP12), which showed successful results on the gel electrophoresis. insert lengths of around 3100 bp’s as expected, and entire plasmids at around 5200bp’s.The plasmids, which were now confirmed both by colony-PCR and restriction cutting, were transformed into our E. coli protein expression strain, ER2566 (SOP14). We then performed SDS-page on the successfully transformed strains (SOP21) and inducing with IPTG. The SDS-page-gels showed no conclusive results. For CPR with overhang oligos N-term 2 and 5, we repeated the PCR (SOP10, step 8.1-8.5), and finally obtained good results by increasing the denaturation time to 20 seconds instead of 10. The CPR-overhang 2 and 5 were then assembled with the corresponding T7-overhang 2 and 5 by overhang-PCR (SOP10, step 8.6-8.8), and bands were observed at expected lengths of ~2500bp’s. We now had T7+RBS+CPR-Nterm 2 (BBa_K4059048) and 5 (BBa_K4059042) with concentrations high enough to cut, which was performed using FastDigest enzymes XbaI and PstI (SOP12). The cut products were ligated into the backbone J61002+J231060+I732820 (SOP13), and the plasmids were transformed into E. coli Top10 (SOP14). Thereafter colony PCR was performed (SOP9). For CPR-Nterm2 (BBa_K4059048), the plates showed no colonies, while single colonies for the CPR-Nterm 5 (BBa_BBa_K4059051) transformation were streak plated the next day. The following colony-PCR showed correct insert lengths for the CPR-Nterm 5 (BBa_BBa_K4059051) transformation. The ON-cultures from successful colonies were used for freeze-stock preparation and plasmid miniprep (SOP4, SOP5 and SOP7).

Week 34 – August 23rd to August 29th
Group PsiDKM
RNA purification was performed, and samples of both not induced and IPTG induced part 5 in ER2566 (BBa_K4059037). After RNA purification, cDNA synthesis was performed form the purified RNA.
Group PsiH:
Cultures of J61002 + J23106 + I732820 with PsiH+Nterm 2 (new) (BBa_K4059039) and PsiH+Nterm 3 (BBa_K4059040) in ER2566 were grown in an overnight culture (SOP4) in later use for qPCR. The cultures were grown to OD = 0.5. Here, 5 mL were removed to function as a negative control for qPCR. The rest of the overnight cultures were then induced with IPTG. The induced cultures were harvested at 1 hour, 2 hours and 3 hours. RNA purification of all the harvested samples for the three different cultures were performed (SOP24), where the RNA qualities were controlled by gel-electrophoresis (SOP8). The gel of J61002 + J23106 + I732820 with PsiH+Nterm 2 (new) (BBa_K4059039) general showed better quality compared to J61002 + J23106 + I732820 with PsiH+Nterm 3 (BBa_K4059040). Here, only very weak bands were seen.
Further, overnight cultures of GroES/EL (BBa_K4059062) and PsiH-Nterm 2 (BBa_K4059039) and CPR-Nterm 5 (BBa_K40590) were made in order to miniprep and send to sequencing (SOP7 and SOP17). In addition, CPR-Nterm 5 (BBa_K4059051) was transformed into ER2566 (SOP14). The transformation showed lots of colonies, and an overnight culture was prepared to make a freeze stock (SOP14 and SOP5).
An overnight culture of ER2566 containing CPR-Nterm 5 (BBa_K4059051) were made for SDS (SOP4). The SDS was prepared for next week (step 7.1 - 7.11 SOP21).
At the end of the week, individual PCR reactions of N-terminal oligo 5, 10 and 11 for PsiH and oligo 33, 34 and 35 for CPR were performed. Oligo 10 and 11 are forward primers to PsiH-Nterm 5 and PsiH-Nterm 6, where oligo 33 is reverse primer to DKM with Deletion for PsiH. Oligo 34 and 35 are forward primers to CPR-Nterm 6 and CPR-Nterm 7.
Group Transport:
This week a new group was formed with the project of trying to get E. Coli to transport the substrate into the cell and transport the end-product out. Amplification and gel purification was made on PsiT1, PsiT2, GFP C-term, and GFP N-term.
Part 2 (Bba_J61002 with Bba_J23106) was minipreped and afterwards cut along with GFP C-term and GFP N-term. Part 2.2 was cut with PstI and XbaI and GFP C- and N-term was cut with XbaI and SpeI. Part 2.2 was ligated together with GFP N-term and GFP C-term. All the ligation products were transformed into Top10 and then plated on AMP.

Week 35 – August 30th to September 5th
Group PsiDKM
Due to trouble getting our part 5 in ER2566 to work properly, it was decided to redo all part making. Biobrick PsiDK and PsiM was put together again to create PsiDKM (BBa_K4059037).
Part 5 was miniprepped from freeze stock and sent in for sequencing.
Group PsiH:
The RNA samples of PsiH+Nterm 2 (new) (BBa_K4059039) and PsiH+Nterm 3 (BBa_K4059040) from week 34 were used for cDNA synthesis (SOP18).
SDS of ER2566 containing CPR-Nterm 5 (BBa_K4059051) was also prepared in the previous week. The SDS was run (SOP21), which showed no bands at all. Therefore, the experiment was repeated but showed the same result.
Additionally, overnight cultures of J61002 + J23106 + I732820 with PsiH+Nterm 2 (old), PsiH+Nterm 2 (new) (BBa_K4059039), PsiH+Nterm 3 (BBa_K4059040), PsiH+Nterm 4 (BBa_K4059036) and CPR+Nterm 5 (BBa_K4059051) were made for miniprep (SOP4 and SOP7).
Two Colony PCRs were also performed with the purpose of sending the samples for sequencing (SOP9). The first colony PCR were made on pSB1C3 + I732820 and pSB1C3 + I732820 with the J61002 backbone. Gel-purification was performed, but the concentrations were too low. Therefore, overnight cultures were prepared and miniprep for sequencing (SOP4 and SOP7).
The second colony PCR were made on GroES/EL (BBa_K4059062), PsiH+Nterm 4 (BBa_K4059036) and CPR+Nterm 5 (BBa_K4059051). Gel-purification was performed on all samples (SOP6) and stored at -20°C before sending them to sequencing the next week (Monday September 6th).
At the end of the week, PCR reactions on several Nterm oligos for CPR, PsiH and DK were performed. 18 different PCR reactions were run (SOP11). No bands were observed for all CPR except CPR-Nterm 15. Also, only DK-Nterm WT showed a band.(SOP6). Bands observed for PsiH-Nterm 8, PsiH-Nterm 9 and PsiH-Nterm “S”. All the seen bands were cut out and purified (SOP6).
Group Transport:
We streaked colonies from the transformation the previous week on plates and checked on a colony PCR. A colony from part 2 (Bba_J61002 with Bba_J23106)+ GFP C-term and part 2 (Bba_J61002 with Bba_J23106) GFP N-term was chosen and observed under a flourescent microscope to see expression of GFP. The two colonies were miniprepped and cut along with T1_F, T2_F, T1_R and T1_R in which the BamHI sites are introduced by specific primers for N-terminal and C-terminal. The cutting was done using BamHI + SpeI and BamHI + NdeI. The cleaved products were ligated with GFP C-term with T1_R and T2_R and GFP N-term with T1_F and T2_F. The ligation products were transformed into Top10. The negative control however grew and AMP plates, colony PCR was used to check the size of the colonies. We discarded the plates due to a negative control that showed colonies.

Week 36 – September 6th to September 12th
Group PsiDKM:
This week prep work was done for qPCR, this included mixing the master mix containing SYBR green master mix 2x, forward, and reverse primers. A total of 6 different primer sets were used, so the expression of different genes could be quantified. The 6 different genes were PsiD, PsiK, PsiM, GyrA housekeeping gene, RpoB housekeeping gene, and T7 polymerase.
The master mix was mixed with the cDNA from week 34 in a 384-well plate. This plate was placed in a qPCR machine. During this week, another transformation was also preformed, here transforming part ? Into Top10. This was tested in a colony PCR.
Group PsiH: In the beginning of this week, the prepared samples from last week (week 35) were sent to sequencing (SOP17). qPCR of the cDNA samples PsiH+Nterm 2 (new) (BBa_K4059039) and PsiH+Nterm 3 (BBa_K4059040) were performed (SOP18). Primer sets for GyrA, Rpo, PsiH and T7 were used. The housekeeping genes GyrA and Rpo are used for normalization of the gene expression of PsiH and T7. The expression of the housekeeping genes should remain constant between the cells and are used for normalization to check errors between the samples.
Furthermore, a new backbone of J61002 containing the medium cons. promoter and LacI (J23106 + I732820) was made, since we still thought there was something wrong with LacI. Overnight culture of J23106 + J61002 and I732820 + pSB1C3 were miniprepped (SOP7). Afterward, J23106 + J61002 was cut with FastDigest enzymes SpeI and PstI and I732820 + pSB1C was cut with the enzymes XbaI and PstI (SOP12). The two parts J23106 + J61002 and I732820 were ligated to create the new backbone J23106 + J61002 + I732820 (SOP13). The new backbone was transformed into Top10 and colony PCR was performed (SOP14 and SOP9) with good results (bands around 1500 Bp’s). An overnight culture of the transformant (SOP4) was made, miniprepped (SOP7) and sent to Eurofins for sequencing (SOP17).
We repeated many of last week’s non-successful PCR-reactions, this time repeated as gradient-PCR’s with different annealing temperatures.
For PsiH we did overhang PCR (SOP11, step 8.1-8.5) with overhang oligo N-term 10 and the overhang oligo meant to introduce a peptide-linker between CPR and PsiH. Both of these overhang-PCR’s were successful with bands at gel-electrophoresis ~1800 bp’s - cut out and purified (SOP 6 + 8). We also assembled T7-overhang Deletion with PsiH-overhang Deletion and T7-overhang “S” with PsiH-overhang WT through overhang-PCR (SOP 11, step 8.6-8.8). The gel-electrophoresis showed bands of correct length of ~1800bp’s, which were cut and purified. This gave us T7+RBS+PsiH-Nterm Deletion (BBa_K4059047) and Nterm WT (J23106+I732820+BBa_K314004).
For the T7 Lac promoter + RBS, we did overhang PCR with overhang oligo Nterm 8, 9, 10, 14, 15 and CPR_Deletion. All worked and showed bands ~160bp except the one with overhang oligo Nterm 15.
For CPR we did overhang PCR with overhang oligos 14 and 15. Overhang oligo 14 worked, with band on gel-electrophoresis at ~2300 bp’s. 15 did not work. We also assembled T7-overhang 2, 14 and Deletion with CPR-overhang 2, 14 and Deletion through overhang PCR (SOP11, step 8.6-8.8). The gel-electrophoresis showed bands of correct length of ~2500bp’s for all three reactions, which were cut and purified. This gave us T7+RBS+CPR-Nterm 2, 14 and Deletion.

Week 37 – September 13th to September 19th
Group PsiDKM
Another attempt at starting over with assembling PsiDKM, but in a backbone containing kanamycin resistence. Here PsiDk and PsiM was amplified using phusion PCR. These were run on a gel and then purified using GFX-gel purification. Phusion PCR was used to assemble PsiDKM, it was then run a gel. The bands were too big; however, it will be sent off to sequencing and the experiment will continue. In order to create a backbone with Kanamycin resistance, a transformation of psb1k3 into top10 was made and called part 14.
Group PsiH:
Part 4.2A is sent for sequencing. Overhang PCR on DKH2, DKH8, DK+H9 and DK+H10. Gave inconclusive results - DMSO might be the issue, amount of DK and H is adjusted for next reactions.
Group Transport
Ligation of C-term + T1_R was made and transformed into Top1, however no colonies except negative control grew. A new transformation was made, and streak plated.
Freezestock 25 and 26 (N+PsiT1 and N+PsiT2) was miniprepped and sent in for sequencing.
Group Knock-out:
Preparation of solutions MgCl2 and CaCl2 of the different molarities, that were needed for the phage experiments. Culture preparation of pCP20, a flippase, and miniprepped it to obtain its plasmid.

Week 38 – September 20th to September 26th
Group PsiDKM
This week, part 4.2 , part 14, and PsiDKM was all cleaved with restriction enzymes, run on gel, and purified. Miniprep was performed of several part 4.2 O/N cultures.
Ligation product was made, in which part 14 and part 15 was used. Hereafter the ligation product was transformed into Top10. The Transformation was plated onto kanamycin plates and then streak plated. A colony PCR was performed on the streak plate colonies. This new part was called part 16.
Group PsiH:
From our old version of the LacI backbone J61002+J23106+I732820, we moved T7+RBS+CPR-Nterm 5 (BBa_K4059042) (which is confirmed by sequencing) to the newest version of the LacI backbone , since the last backbone showed Deletions in LacI in sequencing data. This was done by cutting both the new backbone and the T7+RBS+CPR-Nterm 5 (BBa_K4059051) with the FastDigest enzyme SpeI and the New England Biolabs enzyme BstEII (unique cutter in the middle of LacI), which enabled us to move the last half of the mutated LacI gene (the part AFTER the mutation, therefore unaffected) and the T7+RBS+CPR-Nterm 5 (BBa_K4059051) to the new backbone, where LacI was not mutated. The colony-PCR from this transformation showed good results, indicating that T7+RBS+CPR-Nterm 5 (BBa_K4059051) had been successfully ligated into the new backbone and transformed into Top10. The plasmid was then moved to ER2566.
A gradient overhang PCR on T7 with overhang oligo N-terminal 15 was performed (SOP11, step 8.1-8.5), showing bands at expected length of ~160 bps. We tried multiple times to assemble T7-overhang 15 and CPR-overhang 15 (SOP 11, step 8.6-8.8) with unsatisfying results, but finally found that equal amounts of the two template DNA’s (2ng’s of each) and no DMSO at 60oC annealing temperature worked. DNA purified from gel (SOP6).
We also cut our biobricks T7+RBS+PsiH Nterm 7 (BBa_K4059043), Nterm “S” and Nterm (BBa_K4059047) Deletion and T7+RBS+CPR Nterm 14 (BBa_K4059054) + Nterm-Deletion (BBa_K4059056) using the FastDigest enzymes XbaI and PstI to prepare for ligation with the new LacI backbone (J61002+J23106+I732820), which was cut with SpeI and PstI. The ligation products were transformed into E. Coli Top10 and confirmed by colony-PCR. To make sure of getting constructs without mutations, we also ligated all of the inserts into our initial version of the LacI backbone (from week 26) and transformed these ligation products into Top10 as well. For all of the inserts, at least one colony showed correct length of bands on colony-PCR (~3000bp’s for the PsiH transformants and around 3700 bps for the CPR-transformants). The old versions of T7+RBS+PsiH Nterm 3 (BBa_K4059040), 5 (BBa_K4059042) and 6 (BBa_K4059041) (from week 29) were also cut with XbaI and PstI and ligated into the new LacI backbone (J61002+J23106+I732820), which was then transformed into top 10. These showed good results on the following colony-PCR.
From all of the Top10 transformants confirmed by colony-PCR, plasmids were miniprepped and transformed into our protein expression strain ER2566. We now have the following transformants of ER2566 (all in the backbone J61002+J23106+I732820, either old or newest version): For PsiH:

New LacI-backbone with T7+RBS+H-Nterm 3 (BBa_K4059040)
New LacI-backbone with T7+RBS+H-Nterm 5* (BBa_K4059042)
New LacI-backbone with T7+RBS+H-Nterm 6* (BBa_K4059041)
Old LacI-backbone with T7+RBS+H-Nterm 7* (BBa_K4059043)
New LacI-backbone with T7+RBS+H-Nterm 7* (BBa_K4059043)
Old LacI-backbone with T7+RBS+H-Nterm-WT (J23106+I732820+BBa_K314004)

For CPR::

New LacI-backbone with T7+RBS+CPR-Nterm 5 (BBa_K4059051)
New LacI-backbone with T7+RBS+CPR-Nterm 14 * (BBa_K4059054)
New LacI-backbone with T7+RBS+CPR-Nterm-Deletion (BBa_K4059056)
Old LacI-backbone with T7+RBS+CPR-Nterm-Deletion * (BBa_K4059056)

Group Transport:
We ran colony PCR on the transformants from the previous week and one correct colony of C-term + T1_R was found.
Group Knockout:
We made O/N culture of ER2566 and used to make phage lysate from using P1 phages. We acquired colonies of JW0452 and JW3508 and created phage lysate of these too using P1 phages, in order to knockout the genes acrA and yhjV.
A WT culture of ER2566 was transduced with the P1 lysate of ACRA and YHJV and then plated onto Kanamycin plates. However, due to an error, the wrong plates were used, and normal LA plates were used for plating.

Week 39 – September 27th to October 3rd
Group PsiDKM
The new part 16 was minipreped, cut with SpeI and PstI. The cut part 16 was run on a gel and then purified. The cut part 16 was ligated with PsiDKM. The ligation product of part16 + PsiDKM was transformed into Top10. The transformation was plated on kanamycin plates. The colonies were named part 19.
Group PsiH:
On most of the below listed ER2566 transformants, SDS prep and qPCR-prep was performed with ER2566 Wild Type as negative control. This was done by following SOP21 for SDS-prep. Cells for RNA-purification were harvested from the same cultures, which were induced with IPTG. The only differences were that cells for qPCR was harvested from the cultures at 0H (minus IPTG), at 1 hour (post-induction) AND 2 hours (post-induction), and that the harvested volumes were 1.5mL instead of 1.

For PsiH:

New LacI-backbone with T7+RBS+H-Nterm 5* (BBa_K4059042)
New LacI-backbone with T7+RBS+H-Nterm 6* (BBa_K4059041)
Old LacI-backbone with T7+RBS+H-Nterm 7* (BBa_K4059043)
New LacI-backbone with T7+RBS+H-Nterm 7* (BBa_K4059043)

For CPR:

New LacI-backbone with T7+RBS+CPR-Nterm 14 * (BBa_K4059054)
Old LacI-backbone with T7+RBS+CPR-Nterm-Deletion * (BBa_K4059056)

Cell pellets were stored in -20 freezer overnight.
SDS gels did not show any significant results, may be repeated due to loading of too little of the uninduced (0H) samples.
Group Transport:
Initial production experiments were carried out by us. These involved the old version of PsiDKM in which a deletion of LacI had been detected by Sanger sequencing. We also freeze dryed this.
This week we made preparations for SDS and O/N cultures of Freezestock 25, 26, 39 and 1. The O/N cultures were miniprepped.
Group Knockout:
A new transduction of WT ER2566 with the two phage lysates were made and plated onto 25γ Kanamycin plates. Colonies grew and a colony PCR was performed on YHJV and ACRA. O/N cultures of the colonies were also made, however only YHJV grew in liquid medium and ACRA did not grow. The O/N cultures of YHJV was used for transformation.
The flippase pPC20 was transformed into ER2566 with the knockout of YHJV and then plated onto 25γ Kanamycin plates. The plates were incubated at 30℃ as pPC20 is temperature sensitive. The colonies were run on a colony PCR and results indicated a knockout.

Week 40 – October 4th to October 10th
Group PsiDKM and knockout:
We did Colony PCR on the transformation from the previous week. Multiple of the part 19s were miniprepped and transformed into ER2566 and then plated. Two of the part 19s were chosen and cut with E+PstI, SpeI+PstI and just E. The cut products were run on a gel, but no results were obtained.
Different part 19s from week were transformed into ER2566 containing the YHJV knockout (ER2566 ∆YHJV) and plated on Kanamycin plates.
Part 19 + ER2566 and part 19 + ER2566 ∆YHJV were made and induced with IPTG, cells were harvested at different timepoints after induction. RNA purification was performed on the cells to prepare for qPCR the following week.
Group PsiH:
From last weeks harvested cells RNA purification was performed (SOP24).cDNA synthesis and qPCR were made to investigate the gene expression of the different samples of PsiH and CPR. (SOP18).
At the beginning, both PsiH and CPR were transformed into the backbone J61002+J23106+I732820, which has an ampicillin resistance. However, we need to move CPR over to another backbone with a different resistance to be able to test CPR together with PsiH and GroES/EL in the same bacteria. Also, we want to test CPR with PsiH and PsiDKM. Therefore, CPR was transformed into a backbone with kanamycin resistance (pSB1K3 + J23106 + I732820) and a backbone with chloramphenicol resistance (pSB1C3 + J23106 + I732820). The CPR samples CPR-Nterm-Deletion (BBa_K4059056), CPR-Nterm 5 (BBa_K4059051) and CPR-Nterm 14 (BBa_K4059054) were ligated and transformed with both backbones (SOP13 and SOP14). Colonies from the transformations were streaked out on agar plates. Also, colony PCR and gel-electrophoresis was performed (SOP9 and SOP8). Further, overnight cultures were prepared, miniprepped and freeze stocks were made (SOP4, SOP7 and SOP5). We now had CPR in a backbone with kanamycin resistance and a backbone with chloramphenicol resistance.
In addition, our group made overnight cultures of two different PsiDKM (BBa_K4059037) with kanamycin resistance (SOP4). The samples were miniprepped to make freeze stocks (SOP7 and SOP5). These are later used for transformation in ER2566 to combine PsiH, CPR and DKM. A freeze stock of GroES/EL was made in week 30.
Later in the week, transformations of PsiH-Nterm 3 (BBa_K4059040), 5 (BBa_K4059042), 6 (BBa_K4059041), 7 (BBa_K4059043) and “S” with PsiDKM (BBa_K4059037) in ER2566 were performed (SOP14). The PsiH samples came from miniprep. These transformations succeeded and overnight cultures were made for freeze stocks (SOP4 and SOP5). We had combined PsiDKM and PsiH with different N-terminals in ER2566. All PsiH samples had ampicillin resistance and DKM had kanamycin resistance.
Furthermore, transformations of PsiH-Nterm 3 (BBa_K4059040), 5 (BBa_K4059042), 6 (BBa_K4059041), 7 (BBa_K4059043) and “S” with CPR-Nterm Deletion (BBa_K4059056), 5 (BBa_K4059051) and 14 (BBa_K4059054) in ER2566 were performed (SOP14). The CPR samples came from miniprep. These transformations were also successful and overnight cultures were made for freeze stocks (SOP4 and SOP5). We had combined PsiH and CPR with different N-terminals in ER2566. All PsiH samples had ampicillin resistance and all CPR had kanamycin resistance.
At the end of the week, overnight cultures of the PsiH combined with respectively PsiDKM and CPR were made (SOP4). These cultures were used next week to make triple transformants.
Group Transport:
More production experiments were done. These included both PsiH and PsiDKM. For PsiH tryptamine turned out not to be dissolved when added directly to the media without any other processing. All samples were freeze dried. We didn’t succeed unfortunately due to overload of the freeze dryer.
Preparations for CPR-assay were made, O/N cultures of CPR DEL (4.0), CPR DEL (4.2), CPR 5 (4.2), and CPR 14 (4.2) were diluted and induced with IPTG. Cells were harvested after some time when exponential growth was obtained. The samples were afterwards frozen for later use. Some days later the actual assay was performed, following SOP number 25.

Week 41 – October 11th to October 17th
Group PsiDKM and knockout:
cDNA synthesis were made from the purified RNA from both ER2566 + part 19 and ER2566 ∆YHJV + part 19 and gel electrophoresis were run with the purified RNA.
qPCR was performed using the master mix containing SYBR green master mix 2x, together with forward, and reverse primers. 5 different primer sets were used to quantify the gene expression for PsiD, PsiK, PsiM, GyrA housekeeping gene, and T7 polymerase.
The master mix was mixed with the cDNA from week 40 for both ER2566 + part 19 and ER2566 ∆YHJV + part 19 in two 384-well plate. Both plates were placed in a qPCR machine.
To prepare for LC/MS 100mL cultures containing both ER2566 + part 19 and ER2566 ∆YHJV + part 19 were grown to OD600=0,3 and induced with 4-Hydroxyindol as well as methionine and serine. The cultures were left overnight in a shaking incubator at 37^oC. The cultures were then split into 4x 25mL and placed in 50mL falcon tubes and spun in a centrifuge to separate media from cells. The media were then distributed into 5x 5mL in 15mL falcon tubes and freeze-dried overnight. The cells were transferred to Eppendorf tubes and freeze dried overnight and stored at -80^oC.
Group PsiH:
Your overnight cultures of PsiH-Nterm 3 (BBa_K4059040), 5 (BBa_K4059042), 6 (BBa_K4059041), 7 (BBa_K4059043) and WT (J23106+I732820+BBa_K314004) with CPR-Nterm Deletion (BBa_K4059056), 5 (BBa_K4059051) and 14 (BBa_K4059054) combined with PsiDKM (BBa_K4059037) from last week were used for transformation with CPR-Nterm Deletion (BBa_K4059056), 5 (BBa_K4059051) and 14 (BBa_K405905414 (chloramphenicol resistance) (SOP14). Meanwhile, we also transformed PsiH-Nterm 3, 5, 6, 7 and WT combined with CPR-Nterm Deletion, 5 and 14 together with GroES/EL (SOP14). Both transformations occurred in ER2566.
At first, we thought that our transformation did not work. We chose to incubate our transformation for a longer time, and after 2 days we saw colonies on our triple transformants. For both of our triple transformations, they had only succeeded for PsiH-Nterm 5 (BBa_K4059042), 7 (BBa_K4059043) and WT (J23106+I732820+BBa_K314004). From here, we went on with the transformation of PsiH-Nterm 5, 7 and “S” in both cases.
We made overnight cultures of the successful transformations(SOP4). However, they grow very slowly and it took 2 days before they were grown. In addition, we performed colony PCR and gel-electrophoresis on the triple transformants (SOP9 and SOP8). At first, we used our forward and reverse primers to try to separate PsiH, PsiDKM, CPR and GroES/EL from each other. The expected lengths were PsiH ～3200bp’s, PsiDKM ～5000bp’s, CPR ～3800bp’s and GroES/EL ～3600bp’s. However, we failed to separate fragments from each other and we repeated the colony PCR. For the next colony PCR, we tried to separate the fragments by using specific primer sets for PsiH, PsiDKM, CPR and GroES/EL respectively. We still failed to separate PsiH, CPR and GroES/EL, although we also tried to change the percentage of the gel in gel electrophoresis. This may be because the lengths of the three fragments are very close. We managed to separate PsiH, PsiDKM and CPR for some colonies with the correct lengths. In addition, freeze stocks of the successful triple transformants were made (SOP5).
Group Transport:
Lots of production experiments were performed according to sub. This were done with both single, double and triple transformants. In relation to the solubility problem, it was tested whether dissolving tryptamine in ethanol could be a solution, as it was described in the literature that this method could reach a concentration of 10 mg/ml. This was possible but resulted in the death of the cells when added. Later it turned out that the problem could be overcome by simply dissolving the compound in the medium when this was prepared from the beginning. This should take place at a warm temperature with stirring using a magnet stirrer.

Week 42 – October 18th to October 24th
Group PsiDKM and knockout:
The products from freeze-drying were resuspended in 250µL 50% acetonitril+1% fomic acid solution. These solutions were centrifuged and 150µL supernatant were transferred into micro-insert placed in a vial used in mass spectrometry. To obtain mass spectrometry results the samples were run on Triple Quad LC/MS.
Group PsiH:
In our last week in the lab, we did SDS on several samples of PsiH, CPR and GroES/EL (SOP21). The samples from previous SDS experiments were stored and used for these.
SDS was performed on PsiH+Nterm 5 (BBa_K4059042), 6 (BBa_K4059041) and 7 (BBa_K4059043) with the newest backbone of J61002 + J23106 + I732820. Also, the oldest backbone of PsiH+Nterm 7 (BBa_K4059043) was included.
Further, SDS was performed on CPR+Nterm Deletion (BBa_K4059056) and 14 (BBa_K4059054) with backbone J61002 + J23106 + I732820.
In the end, SDS was made on GroES/EL (BBa_K4059062) with a backbone containing pSB1C3 and BBa_I0500. None of the SDS results showed any results.

Team:SDU-Denmark/Notebook

Notebook

Abstract

Introduction

Protocols