Lab Report II (Biol 203, Spring 2019) NAME (First, LAST):
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 seq
...
Lab Report II (Biol 203, Spring 2019) NAME (First, LAST):
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 GTGA GCGCAA CGCAATTAAT GTGAGTTAGC
121TCACTCATTA GGCACCCCAG GCTTTACACT TTATGCTTCC GGCTCGTATG TTGTGTGG LAC P A (+1) A
181 TTGTGAGCGG ATAACAAT ( Lac O) TT CACACAGGA ( SD) A ACAGCT ATG ( Lac Z#1) A CCATGATTAC GCCAAGCTTG
241 CATGCCTGCA GGTCGACTCT AGAGGATCCC CGGGTACCGG TAGAAAAA AT G ( GFP#1) AGTAAAGGA
301 GAAGAACTTT TCACTGGAGT TGTCCCAATT CTTGTTGAAT TAGATGGTGA TGTTAATGGG
1361 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.
Based on the textbook the plasmid-encoded fusion protein is subject to regulation by cAMP because there is a
CAP binding site thus the cAMP- cap complex is able to bind to the promoter.
HINT: To do this, you will need to consult figures 12.5 and 9.7 in your textbook!
22.
Here is a simple depiction of the pSK+ vector:
[Show More]