iGEM 2019 — Team Tufts

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# Team:Tufts/Notebook

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[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Tufts)

# Tufts iGem Notebook

### September 6th

Lab work begins, we begin the process to electroporate the plasmid into Shewanella

* This process doesn't work
* We read later in  [Hajimorad 2016](https://digital-library.theiet.org/content/journals/10.1049/el.2015.3226) that Shewanella is typically difficult to electroporate

We decide now to use conjugation to insert the plasmid into Shewanella, but first we need to add an OriT site to allow for conjugation.

Trevor runs gibson assembly to insert the OriT site, uses electrophoresis and gel recovery to purify the correctly assembled plasmid. The plasmid is grown in E.coli, then a sample is sent for sequencing to confirm the prescence of the OriT.

### October 6th

The OriT is not found in the plasmid- our hypothesis is that not all plasmids were correctly assembled.

* We ran colony pcr on 4 colonies to check for ones that worked
* These gave us positive results, so then when we sent DNA from these colonies to be sequenced it was confirmed that these plasmids had the correct OriT site.
* Trevor grew a liquid culture from that positive hit, and miniprepped plasmid out of it

### October 13th

We dissolved and filtered DAPI concentrate in water in preperation to grow a rk2 culture that cannot grow without DAPI.

### October 16th

We plated a lawn of rk2 cells

### October 17th

We took the rk2 cells, washed them and electroporated them with the plasmid

* Grew liquid culture from the electroporated cells
* Later in the day we plated the cells on LB + DAPI

### October 18th

Picked colonies from the plate and grew liquid cultures of the rk2, and liquid colonies of Shewanella

### October 19th

Conjugated Shewanella with rk2 strain

* Make two microfuge tubes with 250µl Shewanella + DAPI + LB and 250µ rk2 + DAPI + LB each, as well as one microfuge tube with 250µl Shewanella for control
* Pellet one Shewy+rk2 tube, keep the other in solution
* Incubate the tubes for three hours to allow for conjugation
* Re-pellet all solutions and wash with water to remove DAPI
* Plate half of each Shewy+rk2 tube on one 5-chloramphenicol plate, the other half on a 25-chloramphenicol plate
* Plate the control on a 5-chloremphenicol plate and a 25-chloramphenicol plate
* Let grow for 24 hours

### October 20th

When looking for colonies, found colonies on all 5-chloramphenicol plates including controls, but none on 25-chloramphenicol plates

* Our hypothesis is that Shewanella is able to grow on 5-Chloramphenicol, and that the Shewanella with the plasmid is growing too slowly
* Let grow for another 24 hours

![](https://static.igem.org/mediawiki/2019/4/4c/T--Tufts--dapiLiq.jpeg)

![](https://static.igem.org/mediawiki/2019/3/35/T--Tufts--rk2_culture.jpeg)

![](https://static.igem.org/mediawiki/2019/3/3a/T--Tufts--shewy_col.jpeg)

![](https://static.igem.org/mediawiki/2019/c/cd/T--Tufts--shewy_liquid.jpeg)

### October 21

Inspect our colonies for growth- colonies indicate successful conjugation of plasmid!

![](https://static.igem.org/mediawiki/2019/0/02/T--Tufts--realcolonies.jpg)

Shewanella Colonies

![](https://static.igem.org/mediawiki/2019/f/f5/T--Tufts--control.jpg)

Control plate has no colonies- confirms results


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# Team:Tufts/Experiments

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[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Tufts)

# Experiments

We redesigned and re-made our original design multiple times through the course of our project, this page follows the multiple experiments we performed.

### Experiment 1- Electroporate the MtrB plasmid into Shewanella

* Did not work, we learned through research that Shewanella typically does not respond well to electroporation

### Instead use conjugation to insert plasmid into Shewanella

* Use Gibson Assembly to add the an OriT site onto the plasmid, allowing for conjugation
* Use electrophoresis to filter out the correctly assembled plasmid
* Grow E.coli with the plasmid to ensure it's efficacy in liquid culture, then miniprep the culture to seperate plasmid
* Grow a culture of dapi- E.coli on a plate of LB + Dapi
* Use colonies from the plate to grow a liquid culture of dapi- E.coli
* Simultaneously grow liquid culture of Shewanella
* Combine 250 µl Shewanella and 250 µl dapi- strain in a pellet, similarly 250 µl Shewanella and 250 µl dapi- in a solution, grow for 3 hours
* Re-pellet solution and wash with cold water, then resuspend in LB and plate with chloramphenicol to select for shewanella with the plasmid
* Check to make sure there are colonies on the chloramphenicol plates.

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# Team:Tufts/Results

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[Notebook](https://2019.igem.org/Team%3ATufts/Notebook)
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[Results](https://2019.igem.org/Team%3ATufts/Results)

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[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Tufts)

# Results

### Conjugation of Shewanella

Ultimately we were able to grow a culture of Shewanella with our plasmid

* After conjugation, we found colonies on all 5 chloramphenicol plates, including the non-resistant control. Therefore we could not conclude that our strain had taken up the plasmid
* However after another day of growth, we found colonies on the 25 chloramphenicol plate that we had conjugated in a solution, but not the one that we'd pelleted during conjugation.
* As there are no colonies on the control, we can confirm that our Shewanella took up the plasmid

![](https://static.igem.org/mediawiki/2019/0/02/T--Tufts--realcolonies.jpg)

Transformed Shewanella on Chloramphenicol 25- colonies prove that the plasmid has been taken up

![](https://static.igem.org/mediawiki/2019/f/f5/T--Tufts--control.jpg)

Control plate on Chloramphenicol 25- no growth

![](https://static.igem.org/mediawiki/2019/1/1f/T--Tufts--shewycolonies.jpg)

Shewanella that was conjugated in a pellet- no growth, suggests Shewanella is more effectively conjugated in a solution than a pellet

### Project Achievements

This year we found a project that was both promising, and that excited our team. However due to lack of availability and our teammates' busy schedules, we didn't manage to get as far as we'd liked.

* We managed to design plasmids similar to that used in Cornell 2012 that could be used to detect different analytes
* The plasmid was successfully introduced into a strand of Shewanella through conjugation, after unsuccessful attempts at electroporation.

The continuation of this project is to test the function of these plasmids by measuring the voltages, and further improving the function of this biological sensor.