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 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