7) Fill in the blanks. Don\'t think too hard. Lisa has some e. coli that make GF
ID: 259087 • Letter: 7
Question
7) Fill in the blanks. Don't think too hard. Lisa has some e. coli that make GFP protein only in the presence of arabinose (and do not glow when the e coli are grown in plain LB media with ampicillin). Her friend is skeptical about all of this, and thinks that the non-glowing e. coli don't have the GFP gene, or the GFP protein is being made, but is simply not glowing. a) To show that the non-glowing e. coli still have the GFP gene, Lisa can set up a PCR reaction with primes that recognize the GFP gene, and show that the GFP gene is present in both the glowing e. coli (grown in arabinose media) and the non-glowing bacteria. As a negative control, Lisa should use DNA from e. coli that does not contain the alabinose b) To show the GFP protein is only present when the e. coli are grown in arabinose, she can take some protein from e. coli grown with and without arabinose, and then perform a western blot using antibodies that recognize GFP. AV Which samples should be stained for GFP? The sample in whch the anthi ksillatloch te th poteins ( Protein ii) What is a good positive control sample for Lisa to use in part B? V The sample of glowy E.col grown in arabinose Tron el rabia, RExplanation / Answer
a. To show that the non-glowing E.coli still have the GFP gene, Lisa can set up a PCR reaction with DNA primers that recognize the GFP gene, and show that the GFP gene is present in both the glowing E.coli (grown in arabinose media) and the non-glowing bacteria. As a negative control, Lisa should use DNA from E.coli that does not contain the GFP gene.
(PCR primers are designed specifically to target the gene of interest.
In an experiment, negative control is where no response is expected. Since the E.coli that does not contain the GFP gene cannot be amplified in PCR with the primers specific for GFP gene, it is used as the negative control.)
b.i) The protein samples from both the E.coli that was grown in the presence of arabinose and in the absence of arabinose should be stained for GFP. If the GFP protein is present only when the E.coli are grown in arabinose, only they will be stained positive for GFP and the protein from the E.coli grown without arabinose will be negative for GFP.
ii) GFP protein obtained from E.coli that glows, which was grown in the media containing arabinose, can be used as a good positive control as we know for sure the presence of GFP in it.
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