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Team:UC Davis/Notebook/Week 6
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Most of our progress was halted this week after the discovery that our media was contaminated with some GFP expressing strain. It took us about half of the week to realize the source of our contamination, but now that we have it fixed, we can proceed with construction as planned!
--Monday --
We weren't able to get all the parts for Gibson-ing our construct together. We're still waiting on the 3 repressor PCRs and the pBAD promoter part. The other's have been extracted and have decent concentrations for a Gibson mix.
Today we transformed pBAD+AraC for PCRing tomorrow.
We also proceeded with the construction of our testing plasmid. We digested C0040, C0012, C0051 at XbaI and PstI so that we could ligate it into B0034, which we cut at SpeI and PstI. Additionally, we cut all of our promoter-GFP parts at EcoRI and SpeI, so that we could ligate them with I13453, cut at EcoRI and XbaI. We ran them on a gel, purified them, and plan on ligating and transforming them tomorrow.
We ligated and transformed yesterday's gel purifications today. if this works, we'll be very close to finishing construction!
Strange! Our plates from yesterday are covered with lots of green colonies, even the parts that don't contain GFP! We're going to do some tests; perhaps our media is contaminated, or the water that was used for ligations. We will re-ligate these parts, and re-digest from the minipreps. We ran these re-digestions on gel in order to extract the segment we wanted, but noticed some odd things about the bands. In the lanes containing our vectors, there were three bands where we would expect to only see one. This indicated to us that there was likely some contamination before our digestion. We went ahead and transformed the re-ligated gel purifications from earlier this week.
Took a picture of Nick. He wasn't too happy. We fixed it.
Once again, our plates are contaminated with green colonies. After some deliberation, we believe that our old media was contaminated with GFP-containing bacteria, and that media was used to culture some of our parts prior to a miniprep. Those minipreps, then, have both plasmids. We're going to re-streak the parts from glycerol stocks, and make new minipreps.
We also re-transformed C0051+E0240. Let's hope it works this time.
