You are working at a biotech company and have identified a protein that you and your
colleagues believe inhibits SARS-CoV-2 entry. You wish to clone this gene into a pET
vector (see below).
Previously, you validated the sequence of this gene, which is below (5'-3'; 719
nucleotides in length)
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAG
CTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGC
SATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGC
CCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGCAGTGCTCGC
CCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAA
GCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCC
CGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGG
GCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTA
CAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTG
ACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACT
ACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACAT
GGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCT
GTACAAGTAA
ATG = start codon
TAA = stop codon
1. Using BamH1 and HindIII restriction enzymes,
BamH1
HindIII
describe how you would clone this gene into the
MCS
multiple cloning site (MCS) of a pET vector. The pET
vector MCS has sequences encoding cut sites for
BamH1 and HindIII (Figure 1, note: arrow direction is
5'-3').
antiobiotic
pET
resistance
vector
The recognition / cut sites for BamH1 and HindIII are
gene
shown below (triangles indicate where the restriction
enzyme cuts this sequence).
ori
BamH1
HindIII
Figure 1. pET vector we wish to clone
our gene into.
5'.
GATCC 3'
5' AAGCTT...3"
3' COTAGG... 5'
3' JTCGAA...5"
Specifically:
provide primer sequences you would use to amplify the gene, in preparation for
cloning
describe how you would treat the plasmid DNA and PCR amplicon of the gene
you wish to clone (e.g. treatment with restriction enzymes)
describe how you would clone the gene into pET vector
describe how you would quickly determine if your cloning worked, using agarose
gel electrophoresis, and what you would expect to see if cloning worked as
planned.

Explain the steps involved in producing a homozygous knock-out of a...