Part:BBa_K4800018
Ptrc-sfp-MmCAR(Q302E)-YahK
The promoter was switched to Ptrc, which can be expressed in Escherichia coli KA30, and YciA-sfp-Q302E was derived from BBa_K1655000, an acid-base mutation of carboxylic acid reductase MmCAR at position 302, which replaces glutamine with glutamic acid to improve the efficiency of carboxylic acid reduction, and YahK, an aldehyde reductase.
Build:
Design point mutation primers, clone the whole plasmid by PCR, isolate and purify the product by agarose gel electrophoresis, transfer the copied linearized plasmid into T1 sensory state, apply it on LB solid medium containing 100 μg/mL ampicillin, and incubate it at 37℃ for 12h before selecting a single colony for sequencing by inoculation. The mutated plasmid PRSFDuet-YciA-sfp-Q302E-YahK was obtained.
Test:
Whole-cell catalysis
Through the liquid-phase data, we can clearly see that the catalytic efficiency of the mutated MmCAR is significantly improved compared to the original MmCAR, about 1.5-fold
Fermentation
With the HPLC data, it can be seen that the output of 1,5-pentanediol from the mutated MmCAR is significantly higher than that of the unmutated
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1313
Illegal SpeI site found at 527
Illegal PstI site found at 1748
Illegal PstI site found at 1775
Illegal PstI site found at 3044
Illegal PstI site found at 3071
Illegal PstI site found at 3464
Illegal PstI site found at 3960
Illegal PstI site found at 4604 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1313
Illegal NheI site found at 4872
Illegal SpeI site found at 527
Illegal PstI site found at 1748
Illegal PstI site found at 1775
Illegal PstI site found at 3044
Illegal PstI site found at 3071
Illegal PstI site found at 3464
Illegal PstI site found at 3960
Illegal PstI site found at 4604 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1313
Illegal BglII site found at 2302
Illegal BglII site found at 2928
Illegal BglII site found at 3204
Illegal BglII site found at 3895
Illegal BamHI site found at 1285
Illegal XhoI site found at 4844 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1313
Illegal SpeI site found at 527
Illegal PstI site found at 1748
Illegal PstI site found at 1775
Illegal PstI site found at 3044
Illegal PstI site found at 3071
Illegal PstI site found at 3464
Illegal PstI site found at 3960
Illegal PstI site found at 4604 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1313
Illegal SpeI site found at 527
Illegal PstI site found at 1748
Illegal PstI site found at 1775
Illegal PstI site found at 3044
Illegal PstI site found at 3071
Illegal PstI site found at 3464
Illegal PstI site found at 3960
Illegal PstI site found at 4604
Illegal NgoMIV site found at 3572
Illegal NgoMIV site found at 3634
Illegal NgoMIV site found at 3851
Illegal NgoMIV site found at 4298
Illegal AgeI site found at 1478
Illegal AgeI site found at 2489
Illegal AgeI site found at 2786
Illegal AgeI site found at 3671
Illegal AgeI site found at 3680
Illegal AgeI site found at 4355 - 1000COMPATIBLE WITH RFC[1000]
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