answer c only Tiffany is a research student at the Department of Biosciences of Tunku Abdul Rahman UniversityCollege. Dr. Bane instructs her to prepare chemically competent cells for heat shock transformationfrom old batches of competent cells available in the freezers. The competent cells would eventually beused for the expression of a prokaryotic enzyme using the pET expression vector system.a)Why is DH5a Escherichia coli suitable to propagate the plasmid before protein expression?b)c)During the protein expression step, the plasmids are transformed into a different host cell,BL21(DE3). Explain why this step is pertinent to express the protein of interest.Discuss the steps you would proceed after selecting your E. coli strain to prepare competentcells for heat shock.

Answered: Discuss the steps you would proceed…

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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answer c only

Tiffany is a research student at the Department of Biosciences of Tunku Abdul Rahman University
College. Dr. Bane instructs her to prepare chemically competent cells for heat shock transformation
from old batches of competent cells available in the freezers. The competent cells would eventually be
used for the expression of a prokaryotic enzyme using the pET expression vector system.
a)
Why is DH5a Escherichia coli suitable to propagate the plasmid before protein expression?
b)
c)
During the protein expression step, the plasmids are transformed into a different host cell,
BL21(DE3). Explain why this step is pertinent to express the protein of interest.
Discuss the steps you would proceed after selecting your E. coli strain to prepare competent
cells for heat shock.

Expert Solution

Step 1

c) We can select the E. coli DH5α cells to prepare competent cells for cloning. The steps for the preparation of the chemically competent cells are as follows-

Prepare an LB plate without any antibiotics.
Streak the E. coli cells from the glycerol stock at –80 degrees Celsius and keep it overnight at 37 degrees Celsius.
Pick a single colony, transfer it along the tip to a 4 ml primary culture, and keep it overnight at 37 degrees Celsius.
Transfer 1 ml of the primary culture to 100 ml of the culture in a 500 ml conical flask at 37 degrees Celsius until the OD reaches 0.3 - 0.4.

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