Lab Report II (Biol 203, Spring 2019) NAME (First, LAST): Kabongo, Charlene
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 for
...
Lab Report II (Biol 203, Spring 2019) NAME (First, LAST): Kabongo, Charlene
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 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 TTGTGTGGAA
181 TTGTGAGCGG ATAACAATTT CACACAGGAA ACAGCTATGA CCATGATTAC GCCAAGCTTG
241 CATGCCTGCA GGTCGACTCT AGAGGATCCC CGGGTACCGG TAGAAAAAAT GAGTAAAGGA
301 GAAGAACTTT TCACTGGAGT TGTCCCAATT CTTGTTGAAT TAGATGGTGA TGTTAATGGG
361 CACAAATTTT CTGTCAGTGG AGAGGGTGAA GGTGATGCAA CATACGGAAA ACTTACCCTT
1The 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.
HINT: To do this, you will need to consult figures 12.
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