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Hypothetical lab situation involving transduction and cloning

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#1 luzern



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Posted 02 December 2010 - 05:34 PM

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).

The problem:
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)

Phage Lysate

PHEW! Help with this would be beyond appreciated!

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