2009_iClicker_K_-_Chapter_5

2009_iClicker_K_-_Chapter_5 - 1 Venomous cone snails...

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1) Venomous cone snails produce toxins, like omega conotoxin GVIA that block human calcium channels. We want to produce the conotoxin protein in bacteria. First, we want to amplify the conotoxin sequence (CDS) using the polymerase chain reaction (PCR) , clone it into the vector, replicate the plasmid DNA in bacteria and harvest the plasmid DNA using a DNA plasmid prep . You design sense and antisense primers to amplify the conotoxin in the PCR. On the sense and antisense primers, you incorporate a BamHI restriction site on the end of the sense primer and an EcoRI restriction site on the antisense primer. You amplify the conotoxin CDS in a PCR reaction and clone the gene into the pGEMT-Easy vector . The pGEMT-Easy vector is a TA cloning vector with a “ T overhang ”, that will ligate to the PCR product, with an “ A tail ” left by the Taq DNA polymerase in the PCR reaction. After ligating the insert into the pGEMT cloning vector, you transform the ligation mixture into DH5 alpha bacterial strain by treating the cold chemically competent bacterial cells with a 1 minute heat shock (at 42 C), during which time the plasmid will enter the bacteria. You let the transformation mixture hour of in a 37 C shaking incubator to allow time for the ampicillin resistance gene to express in the bacteria. After one hour, you spread the transformation mixture on a bacterial agar plate containing ampicillin and X-Gal . X-Gal is a colorless substrate the will turn a blue color in the presence of the enzyme beta- galactosidase produced on the LacZ gene . After leaving the bacterial plate in a 37 C incubator overnight, the bacterial plate is shown below. If the conotoxin is successfully cloned into bacteria, which of the following is NOT true? a) The native RNA polymerase on the bacterial chromosome will transcribe the beta-lactamase gene (ie. the antibiotic selection marker) on the plasmid b) There is an ori (origin of replication) on the plasmid for plasmid replication c) The bacterial colonies harboring plasmids with the conotoxin insert will be a blue not a white in color d) All of the following statements (A,B,C) are true CDS of conotoxin PCR amplification of conotoxin BamHI pGEMT vector with T overhang A A EcoRI Amp r ori LacZ T MCS CDS of conotoxin in pGEMT vector ori DH5alpha bacterial strain ligation transformation Amp r pGEMT vector pGEMT vector pGEMT vector
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a) The pGEMT plasmid lacks the neomycin resistance gene for survival in human cells in the presence of neomycin antibiotic b) Transcription of the conotoxin gene in the plasmid requires a mammalian promoter on the plasmid c) Translation requires a mammalian translational initiation site ( Kozak sequence ) just in front of start site d) Expression of the bacterial LacZ gene for turning the human cells blue e) All of the following statements (A,B,C) are true 1) You innoculate a white-colored colony into a liquid culture of bacteria, and carry out a DNA plasmid prep (alkaline lysis / spin column) to isolate purified pGEMT plasmid DNA containing the conotoxin gene insert.
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2009_iClicker_K_-_Chapter_5 - 1 Venomous cone snails...

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