Biology > Lab Report > PCR Cloning and Expression of a GFP fusion protein in E. coli Hunter College, CUNYBIO 203Lab Report (All)

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PCR Cloning and Expression of a GFP fusion protein in E. coli Hunter College, CUNYBIO 203Lab Report II-template

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Lab Report II (Biol 203, Spring 2019) NAME (): PCR Cloning and Expression of a GFP fusion protein in E. coli PART I (No lab data required)- 60 points 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’ end of the GFP coding sequence is as follows: 1 AGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATTCATTAA TGCAGCTGGC 61 ACGACAGGTT TCCCGACTGG AAAGCGGGCA GTGAGCGCAA CGCAATTAAT GTGAGTTAGC 121 TCACTCATTA GGCACCCCAG GCTTTACACT TTATGCTTCC GGCTCGTATG TTGTGTGG(lacP)A(+1)A 181 TTGTGAGCGG ATAACAAT(lacO)TT CACACAGGA(SD)A ACAGCTATG(LacZ#1)A CCATGATTAC GCCAAGCTTG 241 CATGCCTGCA GGTCGACTCT AGAGGATCCC CGGGTACCGG TAGAAAAAAT G(GFP#1)AGTAAAGGA 1301 GAAGAACTTT TCACTGGAGT TGTCCCAATT CTTGTTGAAT TAGATGGTGA TGTTAATGGG 361 CACAAATTTT CTGTCAGTGG AGAGGGTGAA GGTGATGCAA CATACGGAAA ACTTACCCTT The GFP open reading frame was cloned as a BamHI (5’ MCS)/ XhoI (3’MCS) fragment. In this plasmid, GFP is expressed as a fusion protein with LacZ. 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! [Show More]

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