Chen, Joanna HAGOPIAN, RAFFI KRISTOFFERSON, AMY NICOLE SEDOR, DANIEL JOSEPH
Your goal is to determine the activity of transposase and zinc-finger transposase devices. All your constructs will contain a genR cassette flanked by transposon terminal repeats, as well as the self-lysis device. You will induce self-lysis in a buffer containing a plasmid with a pUC origin and ampR gene. You'll determine whether the transposon hops by retransforming the mixture of lysate and plasmid into competent cells and plating on gentamicin and ampicillin medium.
4/15 Team Project Modifications:
You will continue the stability test. At the same time you will be constructing two different plasmids. One plasmid will contain GenR flanked by the Tn5 UTR's on Spec plasmid with an R6K origin. This part will be constructed in a single PCR step using oligos which have been ordered for you. The second plasmid will contain the TN5 transposase downstream of a pBad promoter. You will look for transposase activity by cotransforming the TN5 transposase plasmid with the self-lysis device (p15A/Kan/CAM) and then adding purified GenR plasmid to the lysate. You will transform the lysate into pir- cells and select for Gen.