Difference between revisions of "Part:BBa K4800018"

Latest revision as of 18:47, 6 October 2023

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