In a hypothetical situation I am asked to isolate large amounts of beta-dianase enzyme from human cells.
What is known:
The enzyme is produced by S. cerevisiae, but unstable and degraded in E. coli. An unmapped mutant strain, LS#3, was created that was able to produce large amounts of it.
What is needed:
To isolate the enzyme from human cells using P1 lysate and E. coli because S. cerevisiae shares no homology.
Pretty much need to construct a mutX lacZ∆M15 derivative so one can do blue white screening in the least amount of time (more points for this assignment for the fastest method).
Blue white selection should be used to maximize screening endeavors, but need the unmapped mutX gene from LS#3 in order to stabilize beta-dianase in E. coli. Also, LS #3 cannot complement alpha-peptide from pUC8 plasmids.
E. coli strains
LS#1 = wildtype
LS#2 = ∆lacZ (not alpha acceptor)
LS#3 = ∆lacZ (not alpha acceptor) unmapped mutX (protein stabilizer)
LS#4 = proC::Tn10
LS#5 = lacZ∆M15 (alpha acceptor)
LS#6 = proC
pUC8 (makes alpha-peptide, AmpR)
PHEW! Help with this would be beyond appreciated!
Hypothetical lab situation involving transduction and cloning
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