2810F10Q11ANS - _ _ Your name Your TA's name BioGD2810...

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______________________________ __ _______________________________ Your name Your TA's name BioGD2810 Genetics Fall 2010 Quiz #11 pMAL-p4X is a plasmid vector for use in E. coli which can be used to express and purify large quantities of recombinant proteins; for example, human growth hormone for pharmaceutical purposes. A map of part of the vector is shown on Figure 1; remember that plasmids are circular DNAs. Two genes are illustrated with their directions of transcription as indicated. One of these genes (black) is for tetracycline resistance. The other gene is synthetic and encodes a long fusion protein that has amino acids for (1) maltose binding protein (blue) that can bind to the sugar maltose; (2) the amino acids DDDDK in orange, which constitute a target site for the protease Enterokinase, that can cleave just adjacent to these amino acids; (3) nonfunctional amino acids encoded by a "multiple cloning site" (MCS) that adds restriction enzyme recognition sites to the vector (black); and (4) the lacZ gene of E. coli (red) that encodes the ß-galactosidase protein (required for lactose utilization), but in this case without the initiating methionine. The location of restriction sites for the enzymes EcoRI, HindIII, and SceI are also indicated. Part I: Cloning You want to clone a restriction fragment containing the open reading frame for human growth hormone into the pMAL-p4X vector. You will cut the vector with EcoRI and HindIII and insert the human growth hormone sequences there. (a) (2 points) Explain how you will do a direct selection for E. coli cells containing a pMAL- p4X- related plasmid. Considering just the gene(s) needed for this direct selection, what is the genotype of the E. coli cells that you will use for the transformation? What types of petri plates will you use to select for transformed cells? Tet-S genotype Plate must contain tet plates Note: the plate could be complete (rich) medium, or it could be a minimal medium as long as this medium has a carbon source other than lactose. (b) (2 points) Explain how you will screen by replica plating the transformed E. coli for those that have a plasmid containing an insert. (Hint: this screening requires that the human DNA insert has the entire human growth hormone open reading frame plus a small amount of the 3' UTR.) Considering just the gene(s) needed for this screening, what is the genotype of the
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This note was uploaded on 12/20/2011 for the course BIOGD 6057 taught by Professor Fox during the Spring '09 term at Cornell University (Engineering School).

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2810F10Q11ANS - _ _ Your name Your TA's name BioGD2810...

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