Team:MichiganState/Notebook
Notebooks
Toxin Antitoxin
6/3/21
- Made plates with Kan + DAP, Amp+DAP, Spec + DAP
- Made LB
- Made stock of 30 mM DAP
6/14/21
- Performed Gibson Assembly of pSAC16 gBlocks to get complete pSac16 plasmid
- Performed Gibson Assembly of pFd031 gBlocks to get complete pFd031 plasmid
6/15/21
- Transformed Gibson products into chemically competent E. coli DH5𝜶 via heat shock
6/16/21
- There were no colonies on the transformation plate
- Transformed pSac16 and pFd031 Gibson products into chemically competent E. coli DH5𝜶 via heat shock
6/17/21
- There were no colonies on the transformation plates
- Performed Gibson Assembly for both sets of gBlocks
- Transformed both Gibson products into E. coli WM3064
6/18/21
- Got colonies on both the pSac16 plates and pFd031 plates
- Performed Colony PCR
- Ran gel, there were no bands present for either plasmid
6/21/21
- Transformed both Gibson products into E. coli WM3064 again
6/22/21
- Got colonies on the pFd031 plate
- Performed Colony PCR
6/23/21
- Performed Gibson Assembly again of both the toxin and antitoxin plasmids
- Ran gel of Colony PCR from previous day, no bands present
6/24/21
- Repeated Colony PCR of colonies from the pFd031 transformation plate, no bands were present
- Repeated transformation of Gibson products of pFd031 and pSac16 into WM3064
6/25/21
- Performed Q5 PCR of the pSac16 and pFd031 Gibson productst
- Ran gel, there were no bands present
6/29/21
- Performed Q5 PCR of the Gibson products of both pSac16 and pFd031 again
6/30/21
- Checked concentrations of gBlocks, pFd031 gblocks did not have good peaks
- Ran gel of PCR of the Gibson products, there was a correct band length for pSac16
7/1/21
- Transformed pSac16 Gibson that was confirmed on gel into WM3064
7/6/21
- There were no colonies on the pSac16 transformation plate
- Ordered primers to amplify gBlocks so we don’t run out of them
7/8/21
- Performed Q5 PCR to amplify gBlocks of pFd031 and pSac16 with the primers that arrived
7/12/21
- Ran gel of PCR products of gblocks
- Both pSAC16 gblocks had bands of correct lengths
- No bands for either of the pFD031 gblocks
7/13/21
- Transformation of pSAC16 Gibson product into E. coli WM3064
7/20/21
- Ordered pFD031 vector from Addgene
- Designed new GhoS-GFP block to insert into pFD031
7/21/21
- Performed transformations with various competent cells in lab to test for viability
7/22/21
- Performed PCR cleanup of the amplified pSAC16 gblocks
- Performed SLiCE assembly of the two pSAC16 gblocks
- Transformed into heat competent cells (E. coli WM3064) that were confirmed the previous day
7/22/21
- Prepped pFD031 plasmid from Addgene
7/23/21
- Prepped pFD031 plasmid from Addgene
7/27/21
- Performed MiniPrep of the pFD031 plasmid
7/28/21
- Performed Q% PCR of the PFD031 plasmid to linearize
- Ran gel and correct band size confirmed PCR was successful
- Performed Gibson and SLiCE Assembly of pFD031 and GhoS-GFP gblock
7/29/21
- Transformed Gibson Assembly and SLiCE products into E. coli DH5𝜶
7/30/21
- Made overnight cultures of pFD031 colonies from the transformation
- Performed Colony PCR for six colonies
8/2/21
- Ran gel of Colony PCR products
- Correct band lengths for one of the colonies
- Made overnight cultures of confirmed colony
- Performed SLiCE assembly with the pSAC16 gblocks
8/3/21
- Performed MiniPrep of pFD031-GhoS
- Transformed pFD031-GhoS into E. coli DH5𝜶
- Performed double transformation of pFD031-GhoS and pSAC16 SLiCE products into E. coli DH5𝜶 (added Tetracycline to this plate to induce antitoxin expression)
- Made cryo stocks of E. coli DH5𝜶 with pFD031-GhoS
8/4/21
- Performed Colony PCR from the plate with the double transformation
- Made overnight cultures of E. coli DH5𝜶 with pFD031-GhoS and E. coli DH5𝜶 with pFD031-GhoS and pSAC16-GhoT
8/5/21
- Ran gel for Colony PCR products: did not get bands of correct lengths for either of the assembled plasmids
- Performed new Gibson Assembly with the pSAC16 gblocks
- Transformed pSAC16 Gibson product into E. coli WM3064
- Repeated Colony PCR using a new annealing temperature
- Ran gel of Colony PCR products, was still unsuccessful
8/6/21
- Performed Colony PCR of colonies from the pSAC16-GhoT plate
- Ran gel, but saw no bands present
8/9/21
- Repeated Colony PCR of colonies from the pSAC16-GhoT plate
- Ran gel, still no bands present
- Made overnight cultures of pSAC16-GhoT cells from plate
8/10/21
- There was no growth of overnights
- Transformed the same pSAC16-GhoT Gibson product into E. coli WM306
8/11/21
- Colonies were present on the transformation plates, performed Colony PCR
- Made overnights of colonies
- Ran gel of Colony PCR products, there were no bands present
8/12/21
- There was no growth in the culture tubes
- Performed patch plating of the colonies from the original transformation plate
8/13/21
- The colonies grew on the patch plate
- Made overnight cultures
8/16/21
- There was growth of the overnight cultures
- Performed Colony PCR of the overnights
- Ran gel, there were no bands present
8/30/21
- Made cryostock of overnight culture of WM3064 with pSAC16 plasmid
- Transformed pSac16 into E. coli DH5𝛂
- Made overnight cultures of E. coli DH5𝛂 with pFd031 and WM3064 with pSAC16
8/31/21
- Performed conjugation of WM3064-pSac16 with DH5𝛂-pFd031
- Got colonies for pSac16 transformation into DH5𝛂, made overnight cultures
- Made cryostock of DH5𝛂-pSac16
9/1/21
- Got colonies from the conjugation plate, made overnight cultures (added IPTG to half to induce toxin expression)
- Looked at sequencing results for the pSAC16 plasmid – is not aligning
9/2/21
- There was growth in all overnight cultures from the conjugation plate
- Also growth in all overnight cultures from the conjugation plate with IPTG (indicating the toxin is not being expressed, although the cells were red, so mCherry was being expressed)
- Took OD600 values of all the cultures, cultures with IPTG grew just as well as the cultures without
- Added the different colonies to a 36 well plate, half with IPTG, half without, to try to determine if the toxin is killing cells when induced
9/3/21
- There was growth in all of the cells containing the toxin and antitoxin plasmids in the 36 well plate, with IPTG and without (indicating toxin is not working to kill cells)
- Made overnight cultures WM3064-pSac16 with IPTF to see if cells with only the toxin would not grow when the toxin was induced
9/7/21
- There was still growth in the cells with just the toxin plasmid and IPTG. The culture was red, indicating mCherry was being expressed, however
- Looked more closely at sequencing results for what we thought was the pSac16 plasmid and realized that we accidentally had used another plasmid from lab containing mCherry
- Performed Gibson Assembly of the 2 pSac16 gblocks
- Transformed the Gibson Assembly into WM3064
9/8/21
- Got colonies from the transformation, performed Colony PCR
- Made overnight cultures
9/9/21
- One of the overnight cultures grew
- Ran gel of the colony PCR products , no bands present
- Made more overnight cultures with the culture that grew, added IPTG to half to try to induce toxin expression
9/10/21
- There was growth in all of the culture tubes, but no mCherry expression
- Performed electroporation of pSac16 Gibson product and the pFd031 plasmid into DH5𝛂
- Designed primers to amplify the GhoT-mCherry gene region out of the gblock to insert into another plasmid in lab, pRL814t
- Ordered primers from IDT
9/13/21
- There was growth on the electroporation double transformation plate, made overnight cultures
- Performed Colony PCR
- Ran gel, there were only bands for the pFd031 plasmid, not pSac16
9/14/21
- No growth in overnight cultures from the electroporation
9/15/21
- Performed PCR using Q5 Polymerase to amplify GhoT-mCherry from the gblock
- Ran gel, there was a band the correct size
9/16/21
- Performed a double digest of the plasmid pRL814 with NdeI and HindIII to linearize
- Ran gel of digest, there was only one band indicating the digest was not successful
9/17/21
- Performed two separate restriction digests of pRL814 with each restriction enzyme to determine if one is not working
- Ran gel, got bands for both restriction enzymes, indicating they are both working
- Performed another double digest of pRL814
9/20/21
- Ran gel of the double digest products, still only one band, indicating the plasmid is only being cut once
9/23/21
- Did miniprep of overnight cultures of E. coli with pRL814 to obtain more plasmid
- Performed double digest again
9/24/21
- Ran gel of the double digest, there were 2 bands present indicating the the double digest was successful
9/27/21
- Performed Gibson Assembly of GhoT-mCherry insert (from PCR of gblock) and the linearized pRL814 plasmid
9/28/21
- Transformed the Gibson product into WM3054 via heat shock
9/29/21
- There were colonies on the transformation plate
- Performed Colony PCR
9/30/21
- Ran gel of the Colony PCR products, no bands present
10/1/21
- Made overnights cultures of the colonies from the pRL814-GhoT transformation plate
10/5/21
- There was growth of the overnight cultures
- Miniprepped the overnight cultures to purify what is hopefully the pRL814-GhoT plasmid
HGT Assay (by week)
6/11/21
- Began lab work, our first day we just prepared liquid LB and coordinated with other people in the lab regarding availability of reagents, and had some of our protocols overlooked
- BWe attempted transformations with donor E. Coli/DH5-alpha, with kanamycin resistance, to help prevent contamination. This should yield competent donors and DH5-alpha that is kanamycin resistant
- The transformation did not work, however we are working on fixing what we think went wrong and will attempt this again. Troubleshooting what we think prevented colony formations is helping us learn more and become more proficient in lab
6/18/21
- Attempted transformation of kanamycin resistance
- These colonies were grown overnight in liquid broth which included DAP and kanamycin (one was E. Coli WM6025 and the other was E. Cloni)
- We then used this to make competent glycerol stocks, and they should now be electrocompetent
- 15-30 50uL glycerol stocks of both the donor strain and E. Cloni now stored the freezer
- Lastly, we plated one of the donor strain glycerol stocks on a plate with kanamycin to test if the kanR plasmid was still active
- After checking overnight, a lawn of colonies grew
6/25/21
- Performed three transformations:
- 1. GFP into E. cloni
- To amplify the GFP plasmid
- This was a heat shock transformation done on an ampicillin plate
- Green colonies were seen in UV box, then grown overnight and miniprepped
- 2. GFP into donor strain from glycerol stocks (already has kanR)
- Green colonies formed on plates with ampicillin and kanamycin and grew overnight, glycerol stocks were made
- 3. KanR into C41 (recipient strain)
- Colonies grew on plates with kanamycin, then were grown overnight and stored
7/6/21
- Attempted baseline HGT assay
- We made some mistakes and were unsure about the best way to accomplish certain steps, but have learned what to do differently next time
- No signs of conjugation
- Need to improve positive and negative control
- Possibly an issue regarding OriT
7/19/21
- Worked on testing conjugation of the other plasmid (no oriT) while we waited on the new plasmid (expected 0 colonies)
- Interestingly, with three different ratios of donor to recipient, two of them showed hundreds of colonies (1:1 and 10R:1D) while the 10R:1D plate had 0 colonies
- None of the colonies were expressing GFP
- Only cells that received the GFP/amp plasmid should have survived
- Many things could have caused this, such as not enough antibiotics on the plates, GFP not being expressed, antibiotic resistance developing naturally etc.
- In the long run this is fine since we will not be using that plasmid anyways, but now we know how to do this procedure which we will be repeating this week most likely
- We were not able to receive the plasmid on Thursday/Friday even though we expected to
- Instead we will just get it monday
7/25/21
- Main objectives completed include mini-prepping prl814 plasmid (new GFP plasmid) as well as mini-prepping the plasmid via E. cloni
- The mini-prep did not go perfect, I will discuss this further in the general meeting, but for now what I have should be sufficient
- Cells were also tested with IPTG to ensure there was GFP expression, the cells successfully glowed in each situation
- Glycerol stocks of E. cloni and the donor strain were made
- E. cloni is transformed with the GFP plasmid, this could be used to mini prep if needed in the future
- The donor strain now has kanR and GFP (also spectinomycin resistant) and will be used for the baseline assay
08/02/21
- Began prepping for assay
- Grew glycerol stocks of donor strain w/SPEC and KAN, as well as C41 recipient with KAN
- Retrieved from Micaela’s lab more Spec to makes plates with, then made 25 plates of each of the following
- Spec only
- Spec + DAP
- Spec + Kan
- Spec + Dap + Kan
- Plated E. Cloni w/ GFP for maxi-prep
- For Barrick Lab conjugation test, spun down cells, re-suspended, made 1:1 OD ratio, and plated different dilutions on DAP + Kan plates
- For some reason E. Cloni did not grow so this was attempted again
- Also growing some in IPTG to make sure plasmid is still there
- For the barrick lab conjugation test, scraped up the cells, centrifuged, re-suspended, added IPTG then plated on Spec and Kan (selection plates)
- For main assay, made 1:1 OD ratio of donor:recipient and combined them into one with DAP and kan to grow/conjugate
- Took OD at start time: 0.94
- Was plating hourly, the ODs at the following times were
- Hour 1: 1.05
- Hour 2: 1.10
- Hour 3: 1.26
- Hour 17: 1.36
- Hour 24: 1.66
- I may have had the cap on too tight not allowing enough air until the last 2 measurements
- For the barrick lab protocol, all plates had lawns, and even though IPTG was added, there wasn’t really much glowing at all (Pictures of all this will be shown at the meeting)
- As far as the main assay plates went, for all the plates with DAP that would include the donor; the undiluted, 10, 100, and 1000 fold dilution plates were all very concentrated and only the 1000-fold showed individual colonies (probably too many to count but maybe if I wanted to I could use imageJ)
- From this it may be better to do dilutions of 1000, 2000, 5000, and 10,000
- For the plates that lacked DAP (should only have conjugated colonies),
- At 5:10PM, there are maybe 100-200 colonies undiluted, for 10-fold there are about 57 colonies, at 100-fold only 1, and at 1000-fold 0.
- At 6:00PM, 100-200 colonies undiluted (hard to count) and then none anywhere else
- At 7:00PM, 100-200 colonies undiluted, around 15 at 10-fold, and none afterwards
- 9:00 and 4PM the next day were similar to the others, pictures will be shown tomorrow
- Because IPTG was not added to the selection plates for the main assay, I scraped up cells from each selection plate from yesterday and also cells from the Barrick lab protocol (even though I added IPTG to those, this will be another check), and grew them in broth with IPTG and spec
- I may need to change when and how I add IPTG
- When during conjugation to add it, how much to add to plates, etc.
- Checked on scraped up samples that IPTG was added to
- There is glowing for the most part
- Checked on 17/24hr plates from Friday,
- There wasn't glowing even though IPTG was added
- This may be since IPTG should have been added later (let them conjugate with no IPTG)
- Today I am making 250mL of broth where I'll add E. Cloni w/GFP for maxi prepping
- Adding Spec
- I also added IPTG on top of some of the plates from last week, not expecting anything to happen but maybe Ill see some glowing
Last monday
Tuesday
Wednesday
Thursday
Monday 8/2
08/09/21
- For the transformation assay, again after growing donor and recipient cultures overnight, acquired 1:1 OD ratio of about 1.00, combined and measured the OD over time alongside plating.
- It was difficult to do everything precisely at each hour mark that needed a measurement, since there was a lot of things to do quickly like the dilutions
- Towards the end of the week I checked the plates and colonies grew which was great, and there were in a lot of cases countable colonies that grew when IPTG was added
- Not every plate was countable, as you will see the plates with the donor had too many colonies still, so the experiment will likely need to be repeated
- This time around, I will adjust the dilutions being made for the plate with the donor (up to 100,000-fold), and do a better job at spreading the broth on the plate instead of just dropping it
- Additionally, I plated twice as much because I did duplicates with and without IPTG, and was able to find that adding IPTG did not appear to alter the number of colonies I got
- When I run through this again, IPTG will not be a concern so I will reduce the number of plates by half
- The maxi-prep did not yield me the results I wanted, but luckily this I found out Micaela’s lab has a maxi-prep kit specifically for iGEM that I will be able to use which will make things much easier
- Another test done was a negative control, done on a plate with spec and kan, the three regions that were plated include E. cloni w/GFP, recipient w/kanR, and donor w/kanR & specR
- The recipient did not grow
- E cloni w/GFP grew even though there was kan
- Donor grew even with no DAP
- Unsure why, but may have been too concentrated and perhaps grew on top of one another
- Will test again alongside this week
08/16/21
- Maxi-prep worked much easier using the new kit from Promega
- Obtained a little less that 1.5mL of plasmid, at 57ng/uL
- Made a mistake with the dilutions of the donor and recipient for the assay, they started at a 2,500-fold dilution instead of 25,000
- Too many colonies the first day, but when adjusted for the second day, ended up looking much better
- This time around even though I believe that I did the dilutions for the selection plates right, I kept getting way too many colonies
- Not sure if this is random or what could have gone wrong, I’ll probably need to re-adjust that again, and hopefully get it to stay consistent
- Today (monday), I’m making plates and growing cultures because it seems like I just have to re-do last week unfortunately
- Moving forward, there isn’t much time left in the summer, therefore I think we will create a google slides doc with things we learned or anything similar for the wiki and as a guide for other team members if they want to do anything in the lab when they return