Thursday, August 13, 2009

CLOSE UP of iGEM Calgary's LAB

Hey guys! Summer is coming to a close pretty quick here, and so is iGEM! We are working tremendously hard to try and bring our project to completion! Here’s where we stand as of today (August 12, 2009):

Signaling Circuit

LuxPQ-B0015-R0040-LuxOU-B0015 has been successfully verified in psB1AC3. But that’s old news. We have recently cloned this construct into the pCS26 vector, whose cloning site originally had LuxCDABE flanked by NotI sites. We cut our signaling construct with NotI enzyme, but this does not guarantee that our construct was cloned in the appropriate direction. We transformed the product into TOP10 and XL Gold competent cells, and grew them on Kanamycin plates (pCS26 has resistance for this antibiotic). We have isolated plasmid from eight colonies, and we will verify whether the direction of our construct is correct with a primer that anneals just outside the cloning site on the supposed LuxPQ side. This primer will be paired with a LuxPQ Reverse primer. If we see a PCR product of just over 4kb, we have cloned the construct in the right direction, if we get smearing, however, we have not cloned the product in the right direction.

Moving forward, we require the sigma 70 promoter library that will control the expression levels of LuxPQ in our system. We have had trouble cloning this into the pCS26 vector, and will be trying this again from the start.

Reporter Circuit and Mutants

Pqrr4-B0034-GFP has been successfully verified in psB2K3. Both mutants have been verified in the following constructs in psB1AC3: R0040-B0034-LuxO D47A-B0015 and R0040-B0034-LuxO D47E-B0015. The Pqrr4-B0034-GFP is currently in TOP10 cells, and being made competent once again in order to allow the subsequent transformation of the mutants into the cells. The purpose here is to test the mutant circuits to see if they are working.

Acquiring AI-2

The purpose of constructing all these circuits is to make an AI-2 signaling system. So what else do we need? AI-2! We are currently isolating AI-2 from a species of Salmonella by centrifuging and taking the supernatant that should contain the AI-2. Many controls will be used in order to test whether AI-2 is actually present in the supernatant, namely using Vibrio Harveyi as a response which should glow in the presence of AI-2 because it naturally has the AI-2 signaling system. Once verified that we have AI-2, it can then be used to see if our system responds to AI-2.

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