Biology > Lab Report > Lab Report II (Biol 203, Spring LATEST UPDATE) PCR Cloning and Expression of a GFP fusion protein i (All)

Lab Report II (Biol 203, Spring LATEST UPDATE) PCR Cloning and Expression of a GFP fusion protein in E. coli

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1. Below is a map for a plasmid called ‘pGFP’: The sequence (5’ to 3’) of pGFP containing the promoter region, the 5’ multiple cloning site (with a BamHI site, underlined), and the 5’ e... nd of the GFP coding sequence is as follows: Clearly indicate on the above sequence the following elements: lac Promoter : lacP The start codon for LacZ: LacZ#1 The first codon of the GFP sequence : GFP#1 (here are the first ten amino acids for GFP: MSKGEELFTG) Lac Operator : lacO The transcription start (+1) of the mRNA : circle the nucleotide and indicate +1 The Shine Dalgarno sequence : SD Is the plasmid-encoded fusion protein subject to regulation by cAMP (catabolite repression)? Explain your answer. Yes, It has a CAP binding site which starts from base number 111 and ends at 124 base. So plasmid-encoded fusion protein subject to regulation by cAMP as in presence of the cAMP, cAMP-CAP complex is able to bind the promoter. HINT: To do this, you will need to consult figures 12.5 and 9.7 in your textbook! 2 2. Here is a simple depiction of the pSK+ vector: The vector allows for protein expression in E. coli. Briefly explain the role of the following elements in the context of gene expression and protein production: LacP: is the Lac Operon promoter, a regulatory sequence which contains the -10 and -35 consensus sequences that binds RNA polymerase and start transcription of the lac operon. LacO: is the Lac operator region, a regulatory sequence that the repressor protein binds. Transcription is blocked when the repressor protein is bound to LacO because RNA polymerase cannot bind to the LacP region. LacZ determines whether or not a protein was inserted in the proper orientation. If a protein is in the correct orientation in respect to LacZα then there is a translational fusion. LacZ is a gene that produces beta galactosidase which is an enzyme that breaks down lactose into glucose and galactose. The vector also contains other important elements. Explain their role: AmpR: it is a gene that promote [Show More]

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