Once cloning is complete, a recombinant OXA-M290 protein can be produced in E. coli. Competent E. coli BL21(DE3) cells are transformed with the pET-28a-OXA-M290 plasmid, and the transformed cells are grown on an agar plate that contains kanamycin.  The E. coli BL21(DE3) cells transformed with the pET-28a-OXA-M290 vector are next grown in liquid growth media that contains kanamycin. Once the culture reaches an OD600 of 0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) is added to a final concentration of 0.5 mM to induce protein production.   How does the addition of IPTG result in the transcription of genes that are regulated by the T7 promoter in E. coli BL21(DE3) cells? [4 – 6 sentences suggested]  For Context ONLY: 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 nucleotide sequence for the OXA-M290 gene: >OXA-M290  ATGCGTGTATTAGCCTTATCGGCTGTGTTTTTGGTGGCATCGATT  ATCGGAATGCCTGCGGTAGCAAAGGAATGGCAAGAAAACAAAAGT  TGGAATGCTCACTTTACTGAACATAAATCACAGGGCGTAGTTGTG  CTCTGGAATGAGAATAAGCAGCAAGGATTTACCAATAATCTTAAA  CGGGCGAACCAAGCATTTTTACCCGCATCTAGTGCGAAAATTCCC  AATAGCTTGATCGCCCTCGATTTGGGCGTGGTTAAGGATGAACAC  CAAGTCTTTAAGTGGGATGGACAGACGCGCGATATCGCCACTTGG  AATCGCGATCATAATCTAATCACCGCGATGAAATATTCAGTTGTG  CCTGTTTATCAAGAATTTGCCCGCCAAATTGGCGAGGCACGTATG  AGCAAGATGCTACATGCTTTCGATTATGGTAATGAGGACATTTCG  GGCAATGTAGACAGTTTCTGGCTCGACGGTGGTATTCGAATTTCG  GCCACGGAGCAAATCAGCTTTTTAAGAAAGCTGTATCACAATAAG  TTACACGTATCGGAGCGCAGCCAGCGTATTGTCAAACAAGCCATG  CTGACCGAAGCCAATGGCGACTATATTATTCGGGCTAAAACTGGA  TACGATACTAAGATTGGCTGGTGGGTCGGTTGGGTTGAACTTGAT  GATAATGTGTGGTTTTTTGCGATGAATATGGATATGCCCACATCG  GATGGTTTAGGGCTGCGCCAAGCCATCACAAAAGAAGTGCTCAAA  CAGGAAAAAATTATTCCCTAG

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Once cloning is complete, a recombinant OXA-M290 protein can be produced in E. coli. Competent E. coli BL21(DE3) cells are transformed with the pET-28a-OXA-M290 plasmid, and the transformed cells are grown on an agar plate that contains kanamycin. 

The E. coli BL21(DE3) cells transformed with the pET-28a-OXA-M290 vector are next grown in liquid growth media that contains kanamycin. Once the culture reaches an OD600 of 0.6, isopropyl β-D-1-thiogalactopyranoside (IPTG) is added to a final concentration of 0.5 mM to induce protein production.  

How does the addition of IPTG result in the transcription of genes that are regulated by the T7 promoter in E. coli BL21(DE3) cells? [4 – 6 sentences suggested] 

For Context ONLY:

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 nucleotide sequence for the OXA-M290 gene:

>OXA-M290 
ATGCGTGTATTAGCCTTATCGGCTGTGTTTTTGGTGGCATCGATT 
ATCGGAATGCCTGCGGTAGCAAAGGAATGGCAAGAAAACAAAAGT 
TGGAATGCTCACTTTACTGAACATAAATCACAGGGCGTAGTTGTG 
CTCTGGAATGAGAATAAGCAGCAAGGATTTACCAATAATCTTAAA 
CGGGCGAACCAAGCATTTTTACCCGCATCTAGTGCGAAAATTCCC 
AATAGCTTGATCGCCCTCGATTTGGGCGTGGTTAAGGATGAACAC 
CAAGTCTTTAAGTGGGATGGACAGACGCGCGATATCGCCACTTGG 
AATCGCGATCATAATCTAATCACCGCGATGAAATATTCAGTTGTG 
CCTGTTTATCAAGAATTTGCCCGCCAAATTGGCGAGGCACGTATG 
AGCAAGATGCTACATGCTTTCGATTATGGTAATGAGGACATTTCG 
GGCAATGTAGACAGTTTCTGGCTCGACGGTGGTATTCGAATTTCG 
GCCACGGAGCAAATCAGCTTTTTAAGAAAGCTGTATCACAATAAG 
TTACACGTATCGGAGCGCAGCCAGCGTATTGTCAAACAAGCCATG 
CTGACCGAAGCCAATGGCGACTATATTATTCGGGCTAAAACTGGA 
TACGATACTAAGATTGGCTGGTGGGTCGGTTGGGTTGAACTTGAT 
GATAATGTGTGGTTTTTTGCGATGAATATGGATATGCCCACATCG 
GATGGTTTAGGGCTGCGCCAAGCCATCACAAAAGAAGTGCTCAAA 
CAGGAAAAAATTATTCCCTAG 

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