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 for a plasmid called ‘
...
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 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: *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]
 (1).png)
.png)
