Biology > Lab Report > Hunter College, CUNY-BIOL 203Lab Report II Genetics (All)

Hunter College, CUNY-BIOL 203Lab Report II Genetics

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Lab Report II (Biol 203, Spring 2019) NAME (First, LAST): Sessions 9,10 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 fo... r 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: *Color Coded based on structure above* 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, the plasmid-encoded fusion protein is subject to regulation by cAMP. There is a CAP binding site upstream of the promoter region which allows the cAMP to bind and form the cAMP-CAP complex. HINT: To do this, you will need to consult figures 12.5 and 9.7 in your textbook! 2. Here is a simple depiction of the pSK+ vector: [Show More]

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