Based on the pET-28a map and based on the nucleotide sequence of the OXA-M290 gene, could the restriction enzymes SacI and HindIII be used when cloning the OXA-M290 gene into pET-28a? Why or why not?
For this biology course and based on the results obtained from a previous BLAST search, the bacteria responsible for the infection at a Hospital is most likely a new strain of Escherichia coli. This strain is given the name E. coli AFB.
Earlier, BLAST was used to study a resistance gene from E. coli AFB. These analyses suggest that this strain produces a new β-lactamase, which is given the name OXA-M290. Also, primers were designed to amplify the gene encoding OXA-M290 by PCR.
The next step is to clone the PCR-amplified OXA-M290 gene into an expression vector, so that recombinant OXA-M290 protein can be produced and studied. You will start by going over the process of cloning the OXA-M290 gene into pET-28a. Be sure to reference a plasmid map for pET-28a.
Here is the
>OXA-M290
ATGCGTGTATTAGCCTTATCGGCTGTGTTTTTGGTGGCATCGATT
ATCGGAATGCCTGCGGTAGCAAAGGAATGGCAAGAAAACAAAAGT
TGGAATGCTCACTTTACTGAACATAAATCACAGGGCGTAGTTGTG
CTCTGGAATGAGAATAAGCAGCAAGGATTTACCAATAATCTTAAA
CGGGCGAACCAAGCATTTTTACCCGCATCTAGTGCGAAAATTCCC
AATAGCTTGATCGCCCTCGATTTGGGCGTGGTTAAGGATGAACAC
CAAGTCTTTAAGTGGGATGGACAGACGCGCGATATCGCCACTTGG
AATCGCGATCATAATCTAATCACCGCGATGAAATATTCAGTTGTG
CCTGTTTATCAAGAATTTGCCCGCCAAATTGGCGAGGCACGTATG
AGCAAGATGCTACATGCTTTCGATTATGGTAATGAGGACATTTCG
GGCAATGTAGACAGTTTCTGGCTCGACGGTGGTATTCGAATTTCG
GCCACGGAGCAAATCAGCTTTTTAAGAAAGCTGTATCACAATAAG
TTACACGTATCGGAGCGCAGCCAGCGTATTGTCAAACAAGCCATG
CTGACCGAAGCCAATGGCGACTATATTATTCGGGCTAAAACTGGA
TACGATACTAAGATTGGCTGGTGGGTCGGTTGGGTTGAACTTGAT
GATAATGTGTGGTTTTTTGCGATGAATATGGATATGCCCACATCG
GATGGTTTAGGGCTGCGCCAAGCCATCACAAAAGAAGTGCTCAAA
CAGGAAAAAATTATTCCCTAG
Based on the pET-28a map and based on the nucleotide sequence of the OXA-M290 gene, could the restriction enzymes SacI and HindIII be used when cloning the OXA-M290 gene into pET-28a? Why or why not? A free tool called NEBcutter ( https://nc3.neb.com/NEBcutter/ ) might be useful for answering this question. [3 – 4 sentences suggested]
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