After characterizing the DNA composition of various cats, you identify a protein-coding gene in tigers called stripes and wish to study the structure of the protein product STRIPES. This requires that you purify recombinant ridges from E. coli. First, the stripes gene must be amplified by PCR and then inserted into an appropriate plasmid for bacterial expression. Such a plasmid is diagrammed below. CAP Binding Site ori Ċ Promoter MCS The restriction sites for Aatll and Kpnl are: Aatll 5'-GACGTC-3' Kpnl 5'-GGTACC-3' Laco (Operator) -Kpnl Aatil The coding strand for the stripes gene is shown below, with start and stop codons in bold. 5'-ATGCAACAGTAGCTGAAGCCCAGTGACACCATCGAAAATGTGAAGGCCAAGATGAGGCTCATCTTTGCAGGCAAGCAGCTG GAAGATGGCCGTACTCTTTCTGACTATGCGTCTGAGAGGTGGTATGCAGATCTTCGTGAAGACCCTGACCGGCAAGACCAATGT GAAGGCCAAGATCCAGGATAAAGAAGGCATCCCTCCCGACCAGCAGAGGGCACTCTTTCTGACTACAACATCCAGAAGGAGTCG ACCCTGCACCTGGTCCTGCTGACCGGCAAGACCATCACTCTGGAGGTGGAGCCCAGTGACACCATCGAAAATCCCGACCAGCAG AGGCTCATCTTTGCAGGCAATCACGCACAGTTAA-3' a. List the primer sequences required to amplify the stripes gene with Aatll and Kpnl restriction sites on the appropriate ends such that it can be cloned into the plasmid above for STRIPE expression. Each primer should be 21 bp in length, including the proper restriction site. b. After molecular cloning and confirming you properly cloned stripes into the vector shown, you transform bacteria with the plasmid and select out bacteria that contain the plasmid. Not you want to these transformed bacteria to express the STRIPES. Under what conditions would we need to grow bacteria transformed with this plasmid so that they express STRIPES? Explain your answer, and be sure to describe the important regulatory regions on the plasmid above and what interacts with these regulatory regions in the conditions you have indicated
After characterizing the DNA composition of various cats, you identify a protein-coding gene in tigers called stripes and wish to study the structure of the protein product STRIPES. This requires that you purify recombinant ridges from E. coli. First, the stripes gene must be amplified by PCR and then inserted into an appropriate plasmid for bacterial expression. Such a plasmid is diagrammed below. CAP Binding Site ori Ċ Promoter MCS The restriction sites for Aatll and Kpnl are: Aatll 5'-GACGTC-3' Kpnl 5'-GGTACC-3' Laco (Operator) -Kpnl Aatil The coding strand for the stripes gene is shown below, with start and stop codons in bold. 5'-ATGCAACAGTAGCTGAAGCCCAGTGACACCATCGAAAATGTGAAGGCCAAGATGAGGCTCATCTTTGCAGGCAAGCAGCTG GAAGATGGCCGTACTCTTTCTGACTATGCGTCTGAGAGGTGGTATGCAGATCTTCGTGAAGACCCTGACCGGCAAGACCAATGT GAAGGCCAAGATCCAGGATAAAGAAGGCATCCCTCCCGACCAGCAGAGGGCACTCTTTCTGACTACAACATCCAGAAGGAGTCG ACCCTGCACCTGGTCCTGCTGACCGGCAAGACCATCACTCTGGAGGTGGAGCCCAGTGACACCATCGAAAATCCCGACCAGCAG AGGCTCATCTTTGCAGGCAATCACGCACAGTTAA-3' a. List the primer sequences required to amplify the stripes gene with Aatll and Kpnl restriction sites on the appropriate ends such that it can be cloned into the plasmid above for STRIPE expression. Each primer should be 21 bp in length, including the proper restriction site. b. After molecular cloning and confirming you properly cloned stripes into the vector shown, you transform bacteria with the plasmid and select out bacteria that contain the plasmid. Not you want to these transformed bacteria to express the STRIPES. Under what conditions would we need to grow bacteria transformed with this plasmid so that they express STRIPES? Explain your answer, and be sure to describe the important regulatory regions on the plasmid above and what interacts with these regulatory regions in the conditions you have indicated
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
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Transcribed Image Text:After characterizing the DNA composition of various cats, you identify a protein-coding gene in tigers called stripes and wish to study the structure of the protein product STRIPES. This
requires that you purify recombinant ridges from E. coli. First, the stripes gene must be amplified by PCR and then inserted into an appropriate plasmid for bacterial expression. Such a
plasmid is diagrammed below.
ori
CAP Binding Site
من
Promoter
MCS
The restriction sites for Aatll and Kpnl are:
Aatll 5'-GACGTC-3'
Kpnl = 5'-GGTACC-3'
Laco (Operator)
-Kpnl
Aatll
The coding strand for the stripes gene is shown below, with start and stop codons in bold.
5'-ATGCAACAGTAGCTGAAGCCCAGTGACACCATCGAAAATGTGAAGGCCAAGATGAGGCTCATCTTTGCAGGCAAGCAGCTG
GAAGATGGCCGTACTCTTTCTGACTATGCGTCTGAGAGGTGGTATGCAGATCTTCGTGAAGACCCTGACCGGCAAGACCAATGT
GAAGGCCAAGATCCAGGATAAAGAAGGCATCCCTCCCGACCAGCAGAGGGCACTCTTTCTGACTACAACATCCAGAAGGAGTCG
ACCCTGCACCTGGTCCTGCTGACCGGCAAGACCATCACTCTGGAGGTGGAGCCCAGTGACACCATCGAAAATCCCGACCAGCAG
AGGCTCATCTTTGCAGGCAATCACGCACAGTTAA-3'
a. List the primer sequences required to amplify the stripes gene with Aatll and Kpnl restriction sites on the appropriate ends such that it can be cloned into the plasmid above for STRIPES
expression. Each primer should be 21 bp in length, including the proper restriction site.
b. After molecular cloning and confirming you properly cloned stripes into the vector shown, you transform bacteria with the plasmid and select out bacteria that contain the plasmid. Now,
you want to these transformed bacteria to express the STRIPES. Under what conditions would we need to grow bacteria transformed with this plasmid so that they express STRIPES?
Explain your answer, and be sure to describe the important regulatory regions on the plasmid above and what interacts with these regulatory regions in the conditions you have indicated.
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