34 - MCB 142 Professor Georjana Barnes 11/14/07 Lecture 34...

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11/14/07 Lecture 34 ASUC Lecture Notes Online (formerly Black Lightning) is the only authorized note-taking service at UC Berkeley. Please do not share, copy or illegally distribute these notes. Our non-profit, student-run program depends on your individual subscription for its continued existence. These notes are copyrighted by the University of California and are for your personal use only. Sharing or copying these notes is illegal and could end note taking for this course CTX OPERON What we’re going to do is go over where we stopped last time, which is to see how the researchers came to understand the mechanism behind the ctx operon. What the researchers knew when they started was that there was an operon that expressed two subunits; this was the ctx operon. What they did then was take that operon and put the lacZ coding sequence under the regulatory control of the ctx promoter. Then what they did was look for genes (looking in E.coli) that controlled the expression of the lacZ gene driven by this ctx promoter. To do that, they generated a library of plasmids from a cut-up genome of cholera bacteria and transferred those plasmids into bacteria. The bacteria that they’re working in now is E.coli because this lab strain doesn’t make you sick like cholera. In doing this, they identified the plasmid that turned these E.coli blue; and the reason they were blue was because they were using a X-Gal substrate of beta- galactosidase, which when cleaved turns blue. But not all turn blue, so they separated out the colonies that did turn blue and purified the corresponding plasmid that was turning them blue and that’s how they identified the toxR gene. So they identified in this way the toxR gene and they found it encoded the toxR protein which was a positive regulator of transcription for this operon. Studies of the toxR protein determined that it was a transmembrane protein and that it is monitoring what’s going on outside bacteria. Then they had the toxR gene and wanted to know not only how it regulates the cholera toxin operon but also what other genes this toxR protein might be regulating to give you cholera. To do that experiment, they created a strain of vibrio cholerae that had lacZ coding sequences inserted all over the genome. You can make a huge collection like this and have a library of thousands of strains with this sequence inserted all over the genome. What they did was put the toxR gene under the control of any old constitutive promoter and it’s not regulated, so in these cholera, toxR gene is always expressed. They transformed that plasmid, which will in bacteria express the toxR protein all the time. Then they look for vibrio cholera which would turn blue and they isolate several strains that turned blue out of a collection of inserts. That gave them genes to subclone and identify, so in this way they identified a set of virulence genes. They took those virulence genes and
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34 - MCB 142 Professor Georjana Barnes 11/14/07 Lecture 34...

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