Team:Tufts/Experiments
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.