Lab #3 "Bacterial expression and purification of a designed SARS CoV-2 entry inhibitor"

You are working at a biotech company and have identified a Fab fragment (not a full length antibody) protein that you and your colleagues believe inhibits SARS-CoV-2 entry.

Previously, you described how you:
- sequenced this gene and analyzed the DNA by agarose gel electrophoresis
- cloned this gene into an expression vector

You now wish to express and purify this protein. You are using a pET vector, which uses a T7 promotor upstream of the cloned gene and encodes ß-lactamase as a selection marker. Describe how you would:

a. Select for E. coli that contain this plasmid
b. Induce the expression of your encoded protein SARS CoV-2 drug lead
c. Purify this protein so that you can test it in the lab (not in a human, but in vitro)

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a. The E. coli cells (JM101 strain or another) are transformed by heat-shock (or electroshock) with the recombinant plasmid. The cells are rotated in 500 ml of the medium at 37 °C for a maximum of one hour. Then, the culture is spread on an agar plate that contains the appropriate antibiotic and is placed in the incubator overnight (37 °C). The next day, there is the first screening; the colonies present on the plate have acquired the plasmid and have the antibiotic-resistant gene. These colonies are picked up and grown in 5 ml of the medium at appropriate conditions (temperature, time, etc.). The cells are starved by centrifugation and lysed, and by following a specific protocol, the plasmids are isolated. The plasmids are running on agarose gel (electrophoresis) to confirm the proper purification. Afterward, a few microliters of plasmids, with the appropriate primers, are sent to a company for sequencing. The results must match 100 % with the construct design; if it is the case, the plasmid can be purified at a large scale (1-liter cell culture) and used for protein expression....

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