i need to know if the colonies will grow depending on which site they are being
ID: 186007 • Letter: I
Question
i need to know if the colonies will grow depending on which site they are being inserted? and why?
As part of your summer research project your advisor asks you to clone a protease gene into the plasmid pET2la so that you can subsequently express and purify the protein (note-Proteases degrade proteins!). The plasmid map of pET2la is shown.
pETala() Adapted from Novagen pET Vector table, EMD4Biosciences, EMD4 Millipore USA Note-all of the sites shown on the map occur only once in the vector.
Your lab partner tries to insert the protease open reading frame (ORF) at several different sites in the vector.
a) First, she creates a new construct by inserting the protease ORF at the Pvul I (4428) site. She transforms a suitable cloning strain of E.coli with this construct. Will she see colonies on the appropriate plate the following day? Fully explain your answer.
b) Then, she creates a second construct by inserting the protease ORF at the Kpn I (3229) site. She transforms a suitable cloning strain of E.coli with this construct. Will she see colonies on the appropriate plate the following day? Fully explain your answer.
c) Finally, she creates a third construct by inserting the protease ORF at the Hind III (173) site. She successfully transforms a cloning strain of E.coli with this new construct. She then extracts the new construct DNA from the cloning cells and uses it to transform a suitable expression strain of E.coli. Your advisor tells your lab mate that she has to grow the expression strain containing the construct for a few hours in LB and then add isopropyl-B-galactosidase (IPTG).
Grow for a couple more hours and then harvest the protease protein. Explain why this approch works for expressing a protein such as a protease.
Interpretation Questions 1: Plasmids and Bacterial Transformation I) As part of your summer research project your advisor asks you to clone a protease gene into the plasmid pET2la so that you can subsequently express and purify the protein (note-Proteases degrade proteins!). The plasmid map of pET2la is shown below. 02 pETala() Adapted from Novagen pET Vector table, EMD4Biosciences, EMD4 Millipore USA Note-all of the sites shown on the map occur only once in the vector. Your lab partner tries to insert the protease open reading frame (ORF) at several different sites in the vector. a) First, she creates a new construct by inserting the protease ORF at the Pvul I (4428) site. She transforms a suitable cloning strain of E.coli with this construct. Will she see colonies on the appropriate plate the following day? Fully explain your answer. b) Then, she creates a second construct by inserting the protease ORF at the Kpn I (3229) site. She transforms a suitable cloning strain of E.coli with this construct. Will she see colonies on the appropriate plate the following day? Fully explain your answer. c) Finally, she creates a third construct by inserting the protease ORF at the Hind III (173) site. She successfully transforms a cloning strain of E.coli with this new construct. She then extracts the new construct DNA from the cloning cells and uses it to transform a suitable expression strain of E.coli. Your advisor tells your lab mate that she has to grow the expression strain containing the construct for a few hours in LB and then add isopropyl-B-galactosidase (IPTG).Explanation / Answer
As pvu I is situated at 4428 bp and this site in the selectable marker that provide reistance against the ampicillin because it codes for beta-lactamase enzyme. Once the gene is cloned in this site will not make functionally active beta-lactamase enzyme. Hence she won’t get any colony on ampicillin containing plate. In this vector origin of replication,3227, is present near to Kpn I site 3229, this may destroy the origin of replications site hence vector will not propagate in the E. coli host, hence, after transformation plasmid will not propagate into cell hence it will not provide any resistance marker therefore colonies will not appear on the ampicillin containin plates. In this case where HindIII, 173, site is used than gene is cloned under the T7 promoter, lac operator, ribosomal binding site and translation initiation codon, ATG (NheI and NdeI) , is situated that comes in frame to gene. Hence in this case the gene will express while induction with IPTG in suitable host.
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