iGEM 2021 — Team MichiganState

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# Team:MichiganState/Notebook

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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

*Last monday*

+ Began prepping for assay
+ Grew glycerol stocks of donor strain w/SPEC and KAN, as well as C41 recipient with KAN

*Tuesday*

+ 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

*Wednesday*

+ 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*

*Thursday*

+ 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.

*Monday 8/2*

+ 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

### 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

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# Team:MichiganState/Engineering

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Design

# Engineering Success

## Research

Michigan State iGEM’s research begins with questions regarding the three layers of our Comprehensive Biocontainment System, **Synterception**:

1. How has GhOST been characterized and how is it an effective Toxin-Antitoxin system
2. How can we characterize and target foreign conjugative plasmids?
3. How efficiently can Synterception remediate baseline conjugation?

|  | Toxin Antitoxin System | Baseline Conjugation Assay and Modeling |
| --- | --- | --- |
| **Visualize** |  |  |  |
| **Design** | To characterize the GhoST system, we wanted to create a two vector system to determine the relative levels of expression of GhoT that would kill the cells, as well as the amount of GhoS that would “rescue” the cells and allow them to grow in the presence of GhoT. We decided to use two vectors developed by Dr. Jonathan Silberg’s lab, pSac16 and pFd031 [1], which are two compatible vectors with different inducible promoters. We could then separately induce GhoT and GhoS in the same cell transformed with both vectors. We first designed two gBlocks for each vector that when assembled together, would form the complete plasmid. One of the gBlocks for pSac16 was the GhoT toxin fused to mCherry, and on one of the gBlocks for pFd031 was the GhoS antitoxin fused to GFP. We decided to attach fluorescent markers to the toxin and antitoxin to be able to qualitatively measure the levels of expression of both proteins. This way, we could determine how much GhoT expression was needed to kill the cells, as well as how much GhoS expression relative to GhoT was needed to allow the cells to grow. For each vector, we designed one of the gBlocks to have homologous overhangs to the other gBlock, so Gibson Assembly could be used to form the complete plasmid. However, this design method was unsuccessful in generating the assembled plasmids, so we later ordered the pFd031 and pSac16 plasmids from Addgene and designed new gblocks to insert into them, in a more traditional cloning style. |  |
| **Build** | Our initial design with the two gBlocks did not work to assemble the vectors. Once we decided to order the full plasmids from Addgene, we amplified the backbone of the pFd031 plasmid and then performed Gibson Assembly with the new gBlock containing the GhoS gene fused to GFP. We then transformed our plasmid into heat competent E. coli DH5⍺. In the end, we were only able to build the antitoxin vector and transform it into our cells. |  |
| **Test** | Unfortunately, we were unable to test our constructs, as the assembly of the vectors was never completed. We planned to characterize the GhoST system by having E. coli containing the GhoT vector, the GhoS vector, and cells that had both vectors present. We planned to add varying levels of the inducers of both the toxin and antitoxin to the different cells in a plate reader. We would measure the OD600 to determine the cells’ death due to the toxin, as well as the growth allowed when “rescued” by the antitoxin. We would also measure the fluorescence produced, as this was indicative of both GhoT and GhoS expression, to determine the relative levels of the toxin that would induce death, as well as the levels of the antitoxin that would inhibit the toxin’s affects. | In order to test how far to dilute these cultures, plates were divided into 12 sections ranging anywhere from being undiluted, up to 150,000-fold, in order to find what dilutions were necessary to get a countable amount of colonies. |
| **Learn** | We learned during our time in the lab that our design of two gBlocks to assemble a vector was not working. We were unsuccessful in all of our Gibson Assembly attempts, so we first tried to use another method to assemble the gBlocks together called SLiCE ligation. Unfortunately, we were not able to get this to assemble our vectors either. So, we realized that we had to change our design. We ordered the plasmids Pfd031 and pSac16 from Addgene and designed new gBlocks containing the toxin and antitoxin both fused to their respective fluorescence proteins. This new approach allowed us to obtain the fully assembled pFd031 antitoxin plasmid | **Ratio of D:R**  We found out that the ratio of donor to recipient was not a critical factor when testing conjugation. **Dilutions for transconjugants**  Although testing out a large scale of dilutions for each of our plates was very time consuming, it was crucial for obtaining measurable results and provided us with insight with regards to the amount of transconjugants that will from with a given concentration of donor and recipient cells. |
| **Improve** |  |  |
| **Research** |  |  |

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# Team:MichiganState/Experiments

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Experiments and Protocols

# Experimental Protocols

## Prepatory Protocols

### Heat Shock Transformation

1. Thaw heat competent cells on ice
2. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 50 μL of cells in a microcentrifuge tube, gently mix by flicking
3. Leave cells on ice for 30 minutes
4. Heat shock at 42 °C for 30 seconds
5. Place tubes back on ice for 2 minutes
6. Add 250 μL of SOC media and 3 µL DAP
7. Incubate the cells at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate
8. Warm selection plates to 37°C
9. Remove 20 μL of cells and plate for a low concentration
10. Spin cells down for 2 minutes at 8,000 rpm for 2 minutes
11. Remove most of the supernatant by either decanting or pipetting
12. Resuspend the cell pellet in the remaining supernatant (80-100 µl) and plate
    Incubate overnight at 37°C

### Electroporation Transformation

1. Place the following materials on ice: Electroporation cuvettes, (thawed) plasmid DNA, and a conical tube with sterile LB. Label Eppendorf tubes for each transformation.
2. When everything else is ready, get the electrocompetent cells (50 µL aliquots) out of the freezer and let them thaw on ice.
3. Add plasmid DNA (1-5 µL) to the cells.
4. With a pipette set at 50 µL, pipette up and down once to mix.
5. Then (using the same tip), pipette all 50 µL into an electroporation cuvette.
6. Electroporate at 1800 V and record the time constant.
7. Immediately add 1 mL of cold LB to the cuvette.
8. Decant the LB/cell mixture into a labelled Eppendorf tube.
9. Recover at 37°C for 1.5 hours.
10. Centrifuge cells for 1 minute at 15,000 rpm.
11. Remove most of the supernatant by either decanting or pipetting.
12. Resuspend the cell pellet in the remaining supernatant (80-100 µl) and plate.
13. Incubate overnight at 37°C.

### Conjugative Baseline Assay

![](https://static.igem.org/mediawiki/2021/8/8f/T--MichiganState--HGTAssay.svg)

:   ***Day 0***

1. Transform E. coli WM6026 with GFP plasmid. Grow overnight on LB agar plate +0.3mM DAP + Amp

:   ***Day 1***

1. Pick colonies from the GFP donor strain plate that are expressing GFP
2. Grow E. coli WM6026 with GFP plasmid overnight, shaking with antibiotic (Amp) and DAP (0.3 mM). Grow in LB. Grow recipient strain E. coli C41 overnight in LB

:   ***Day 2***

1. Spin down 3824g 5 min, wash both strains in 1 mL LB +DAP+ (3x)
2. Combine the 2 suspensions in a 20 mL 1:1 OD ratio in a microfuge tube, pipette to mix
3. Take OD600 measurements of both cultures (aim for OD of 1.0)
4. Add 20mL suspension to 50mL conical tube). Cap and place in shaking incubator for 24 hours
5. Also grow 10mL of each culture in individual 50mL conical tubes, take OD600 measurements at each time intervals, plate recipient sample on Kan in same way as below for as many timepoints as possible
6. Taking samples: start culture at 3:00 pm, take samples at 4:00 pm, 6:00 pm, 8:00 pm, 10:00 am, 3:00 pm
7. Take 1.5 mL of the culture and wash with LB (Spin down at 3824g for 5 minutes)
8. Measure OD600 of 1mL in the spectrophotometer
9. Section plate into quarters
10. Perform serial dilution. In quadrant 1, plate 25uL of undiluted culture. Perform 3 serial dilutions, 1:10 each time (undiluted, 1:10, 1:100, 1:1000) . Plate 25uL of the dilution in the appropriate quadrant
11. Label each plate with the dilution, what time the sample was taken, and plate type
12. Plate each sample on Kan+Amp, DAP+Kan+Amp
13. For the controls, plate recipient on DAP+Kan+Amp once, plate recipient on Amp-only once

:   ***Day 3***

1. Perform a luminescent screen and record the number of colonies expressing GFP on each plate
2. Record the total number of colonies on each plate

## Synterception Protocols

### Lysis Assay and Establishing a Standard Curve

1. Transform recipient E. coli strain with GFP plasmid (as you would for the baseline assay). Grow in 250mL LB broth overnight at 37°C
2. Grow untransformed recipient strain in 250 mL LB broth overnight
3. Grow untransformed recipient strain in 250 mL LB broth overnight
4. Remove 200 mL broth from each culture. Perform 10 serial dilutions (by a factor of 2), such that each dilution has a final volume of 30mL
5. Lyse each dilution using a french press
6. Centrifuge for 20 minutes at 2000xg to pellet cell debris in each sample
7. For each dilution, remove 1 mL of the supernatant and place in a cuvette. Quantitate GFP fluorescence with spectrophotometer
8. Remove 1 mL of broth from each culture. Perform 5 serial dilutions (by a factor of 2) and plate on LB agar, incubate overnight
:   (This is to establish a concentration in CFUs/mL of the original culture, in order to determine how many cells were initially lysed)
9. Plot the number of cells lysed vs. GFP fluorescence
10. Perform basic HGT assay. On day 2 before centrifugation, extract 1.5mL of broth from the large culture and spin down in an Eppendorf tube for 20 minutes at 2000xg. Transfer supernatant to cuvette and quantitate fluorescence in supernatant using spectrophotometer. Proceed with assay.

### Transformation Assay

:   *Day 1*

1. Grow E. coli overnight, shaking in LB at 37°C

:   *Day 2*

1. Remove 50 mL of culture in a 50mL conical tube and centrifuge 20 minutes at ~13,000xg or max speed
2. Remove supernatant. Wash with PBS and centrifuge again
3. Remove supernatant. Resuspend pellet in 10mL LB
4. Repeat this 7 times to get 7 different tubes.
5. Add:

1. Tube 1 : No plasmid (Control)
2. Tube 2 : Undiluted plasmid (~50ng/µL)
3. Tube 3 : 1/75 dilution
4. Tube 4: 1/100 dilution
5. Tube 5: 1/200 dilution
6. Tube 6: 1/500 dilution
7. Tube 7: 1/1000 dilution

6. Plate 100 uL of each dilution onto an LB plate. Plate 100 uL of each dilution onto an antibiotic plate. Incubate for 8-12 hours

:   *Day 3*

1. Record the total number of colonies on each plate. To calculate the transformation frequency: (Number of Antibiotic CFUs)/(Total CFUs on LB)

### Gibson Assembly

1. Calculate the optimal amounts of DNA for assembly: pmols =(weight in ng)x1000/ (base pairs x 650 daltons). Use the NEB BioCalculator to determine amounts of backbone and insert to add.
2. Add x μl of backbone to the PCR tube
3. Add x μl of the insert to the PCR tube
4. Add x μl of NEB HiFi DNA Assembly Mix to the PCR tube (1:1 ratio of Assembly mix to total DNA)
5. Incubate the reaction for 15 minutes at 50 ºC

### SPLiCE Ligation

1. Calculate the optimal amounts of DNA for assembly: pmols =(weight in ng)x1000/ (base pairs x 650 daltons). Use the NEB BioCalculator to determine amounts of backbone and insert to add.
2. Add x μl of backbone to the PCR tube
3. Add x μl of the insert to the PCR tube
4. Add 1 μl of SLiCE buffer to the PCR tube
5. Add 1 μl of SLiCE extract to the PCR tube
6. Add nuclease free water to get 10 μl reaction
7. Incubate for 15 minutes at 37 ºC

### Colony PCR

1. Place 50 μl of sterile water in as many PCR tubes as colonies that are being screened.
2. Pick colonies and place in corresponding water PCR tube, pipette up and down to mix.
3. Set up a reaction as follows (per tube, so can be scaled up):

* 5 μl MasterMix
* 3 μl H2O
* 0.5 μl forward primer
* 0.5 μl reverse primer
* 1 μl of diluted cells

4. Use NEB Tm Calculator to determine the correct annealing temperature for the primers used in the PCR. Calculate the extension time as 30 seconds per kB.
5. Run the PCR reaction in the thermocycler

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# Team:MichiganState/Results

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Results and Analysis

# Results

While the broader experimental goals include multiple genetic interventions to restrict transfer of recombinant DNA (see [Project Description](https://2021.igem.org/Team%3AMichiganState/Description)), laboratory access was restricted due to COVID-related health considerations. We therefore focus our description of the experimentation of the Synterception project on the  **Conjugative Baseline Assay**  and  **Computational Results** , as these were the best developed experimental goals during the time period.

## Conjugative Baseline Assay Evaluating the efficacy of interventions designed to reduce DNA transfer requires estimations of the rates of background transfer events. The experiments below were conducted to establish rates of DNA exchange from a donor to recipient cells and to estimate the likely mechanisms of transfer (e.g., conjugation, uptake of extracellular DNA, etc.) In doing so, the following experimental groups consisted of liquid cultures of bacteria: donor control, *E. coli* WM3064, recipient control, *E. coli* C41, and a 1:1 ratio mixture of the donor and recipient strains. *Figure 1* Donor Control Colonies counted on petri dish containing DAP | Dilution Fold | t0 start | t1 2 hours | t2 5 hours | | --- | --- | --- | --- | | 150000 | 31 | 70 | 29 | | 125000 | 31 | 83 | 37 | | 100000 | 39 | 89 | 55 | | 75000 | 56 | 88 | 63 | | 50000 | 65 | 95 | 84 | | 25000 | - | - | 95 | | | | | | | --- | --- | --- | --- | | Average | 3.98 \* 106 | 8.23 \* 106 | 4.68 \* 106 | | Standard Deviation | 5.12 \* 105 | 2.50 \* 106 | 5.04 \* 105 | The Donor Control colonies consist of *E. coli* WM3064 (DAP auxotroph) containing GFP, a reporter acting as the gene of interest for the purpose of these Baseline assays. Cells were plated on appropriate selection plates in order to determine the basal rates of colony forming units (CFUs). These results were collected independent from the conjugative mixture data. The purpose of this trial was to quantify the colonies corresponding to the OD600 to better understand the donor strain’s presence in the conjugative mixture. *Figure 2* Recipient Control Colonies counted on petri dish containing Ampicilllin | Dilution Fold | t0 start | t1 2 hours | t2 5 hours | | --- | --- | --- | --- | | 150000 | 37 | 47 | 60 | | | | | 125000 | 37 | 54 | 71 | | | | 100000 | 46 | 83 | 89 | | | | 75000 | 59 | 75 | 95 | | | | 50000 | - | - | - | | | | 25000 | - | - | - | | | | | | | | | --- | --- | --- | --- | | Average | 4.08 \* 106 | 6.93 \* 106 | 8.48 \* 106 | | Standard Deviation | 5.08 \* 105 | 1.10 \* 106 | 9.02 \* 105 | Likewise, the recipient control group consisted only of the E. coli C41 strain with a selective marker. The colonies recorded along with the OD600 were used to equate the recipient strain grown in the conjugative mixture. *Figure 3* Conjugative Mixture Colonies counted on petri dish containing DAP | Dilution Fold | t0 start | t1 2 hours | t2 5 hours | | --- | --- | --- | --- | | 150000 | 11 | 25 | 22 | | | | | 125000 | 13 | 31 | 27 | | | | 100000 | 19 | 29 | 36 | | | | 75000 | 22 | 42 | 52 | | | | 50000 | 33 | 43 | 68 | | | | 25000 | 57 | - | - | | | | | | | | | --- | --- | --- | --- | | Average | 1.64 \* 106 | 3.23 \* 106 | 3.49 \* 106 | | Standard Deviation | 1.25 \* 104 | 7.87 \* 105 | 2.74 \* 105 | The Conjugative Mixture colonies collected above, represent the total colonies (donor and recipient strains) from a liquid culture. The divided colonies are seen in Figure ### Ratio of Colonies. The colonies were split based on the control groups and OD600 from the corresponding strains and mixture data. *Figure 4* Transconjugant Colonies Colonies counted on petri dish containing Ampicillin | Dilution Fold | t0 start | t1 2 hours | t2 5 hours | | --- | --- | --- | --- | | 10000 | - | - | - | | | | | 1000 | - | - | - | | | | 500 | - | - | - | | | | 100 | 9 | 40 | 36 | | | | 50 | - | - | - | | | | 25 | - | - | - | | | | | | | | | --- | --- | --- | --- | | Average | 9.00 \* 102 | 4.00 \* 103 | 3.60 \* 103 | The Conjugative Mixture colonies collected above, represent the total colonies (donor and recipient strains) from a liquid culture. The divided colonies are seen in Figure ### Ratio of Colonies. The colonies were split based on the control groups and OD600 from the corresponding strains and mixture data. *Figure 5* OD600 Colonies counted on petri dish containing Ampicillin | Strain | t0 start | t1 2 hours | t2 5 hours | | --- | --- | --- | --- | | Donor Control | 0.960 | 1.20 | 1.35 | | | | | Recipient Control | 0.950 | 1.12 | 1.23 | | | | Conjugative Mixture | 0.955 | 1.22 | 1.43 | | | The OD600s from the corresponding colony data collection were recorded. The conjugative mixture received a 1:1 volume ratio of both the donor and recipient controls. Multiple Sequence Alignment With the use of bioinformatics techniques the team was able to perform a gene homology assessment on plasmids in the Incompatibility P family. Addressing primarily the homology of the TraI gene, which codes for the conjugative relaxase, an enzyme essential for the process of conjugation and DNA transfer. The catalytic center of the relaxase, Tyrosine 22 was discovered, and created a list of 10 sequences Cas9 could target to knock out the TraI gene at this catalytic tyrosine. Our consensus sequence is as follows: AAGGATAATTACTATGTGCTGGG Another 9 sequences have been arbitrarily selected from the same multiple sequence alignment from which the consensus sequence was derived. The nucleotide sequences are as follows: AAAGACAATTACTACGTCATCGG AAGGATAATTACTACGTCATAGG AGGGATAACTACTACGTGCTGGG AAGGATAATTACTATGTACTGGG TCCGATAACTACTATTTTCTGGG AAGGATAATTACTATGTCTTGGG AAAGATAATTACTACGTCATCGG GAAGACAACTACTATGCCAGCGG AAGGATAATTACTACGTTATCGG With this nucleotide region knocked out, the relaxase should cease to function. These sequences in the IncP conjugative plasmids play a crucial role in the functionality of the TraI gene. The selected sequences were chosen arbitrarily from a dataset composed of 284 IncP plasmid sequences. Due to time constraints, a more rigorous selection process was not chosen to determine which 23 base pair sequences we would include as targets for Cas9. Horizontal Gene Transfer Kinetics Model Above, the plot is a result of a Python script using End-Point and Markov Method inspired modeling. The initialization of the script begins with two cells: one donor (pink) and one recipient (green). While the program calculates the states over time, the occurrences in conjugated cells (brown) and transconjugants (blue) change accordingly. As a tool for simulating the steady states for baseline conjugation occurring (respective to the Conjugative Baseline Assay), the slider pictured above the plot is adjustable in the program. The applied values on the slider correspond to the probability of transitioning cell states. Described below, the probabilities are defined by the mathematical and biological possibilities of transformation. Analysis The objective of the **Conjugative Baseline Assay** was to determine the rate of conjugation and transformation occurring from an approximate 1:1 ratio of donor to recipient cells. *Figure 6* Ratio of Colonies | Strain | t0 start | t1 2 hours | t2 5 hours | | --- | --- | --- | --- | | Conjugative Mixture | 1.64\*106 | 3.23\*106 | 3.49\*106 | | | | | Donor in Mix | 8.26\*105 | 1.59\*106 | 1.65\*106 | | | | Recipient in Mix | 8.18\*105 | 1.48\*106 | 1.50\*106 | | | | Calculated Sum (D+R) | 1.64\*106 | 3.07\*106 | 3.15\*106 | | | | Standard Error | 0.00 | 7.95\*104 | 1.17\*105 | | | | Percent Error | 0.00% | 4.92% | 9.79% | | | The following Recipient and Donor in Mix colonies were calculated using the colonies present in the respective controls and OD600. An adjustment ratio was used to isolate the relationship between the OD600 and the colony count from the control groups. The strain in the mix was determined by half the OD600 divided by the adjustment ratio; this provided ratios for transconjugants and each strain in the mix. *Figure 7* Ratio of Transconjugants *Figure 6* Ratio of Colonies | Strain | t0 start | t1 2 hours | t2 5 hours | | --- | --- | --- | --- | | D:R Ratio | 1.01 | 1.07 | 1.10 | | D:T Ratio | 9.18\*102 | 3.97\*102 | 4.58\*102 | | R:T Ratio | 9.08\*102 | 3.71\*102 | 4.17\*102 | | R:T Ratio | 1.12\*10-3 | 2.68\*10-4 | 3.05\*10-4 | The ratios in the table above were used to describe the rates of conjugation (Donor to Transconjuant) and rates of transformation (Recipient to Transconjugant).

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