Part:BBa_M50052:Experience

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Applications of BBa_M50052

In conjunction, BBa_M50004 (containing alkB1 protein, ABO_2707) and Ba_M50005 (containing alkG protein, ABO_2708) are meant to degrade alkanes measuring up to thirty-two carbons as the first step of the digestive process of marine bacterium Alcanivorax borkumensis. Specifically, these two enzymes add an -OH group onto the alkane. BBa_M500052 isolates these two proteins to facilitate their transformation into E. coli and other bacteria in an attempt to replicate this degradative behavior in more controlled environments.

User Reviews

Stanford BioE44 "Team WERK" 12.10.2016
We transformed E. coli with both BBa_M50004 and BBa_M50005 in an attempt to replicate the first step of A. borkumensis' alkane degradation in different bacteria. Under proper induction (IPTG and rhamnose, respectively), our Western blot protocol revealed moderate levels of alkB1 production and high levels of alkG production. However, pH testing failed to confirm the cooperative function of alkB1 and alkG, with transformed bacteria in alkane-rich conditions behaving relatively indistinguishably from those in standard conditions. This may have been due to a failure in double transformation, with the two proteins being expressed at disproportionate levels.

The above images show the results of our Western Blot protocol (using anti-Flag antibody and anti-His antibody to reveal alkB1 [left] and alkG [right] production, respectively). In both cases, singly-transformed bacteria (highlighted regions) demonstrated greater protein expression than doubly-transformed bacteria, though doubly-transformed bacteria did demonstrate reasonable production of alkG.

The above graph shows the results of our pH testing; each dataset represents a different instance of transformed E. coli. Measurements were normalized against the initial pH of each bacteria culture. Under proper collaborative function of the alkG and alkB1 enzymes, pH ought to have decreased over time relative to the control; however, no concrete results were observed.

UNIQ80db92784d4a15cc-partinfo-00000000-QINU UNIQ80db92784d4a15cc-partinfo-00000001-QINU

Stanford Location

Plasmid name: ABO_2707 + ABO_2708
DNA2.0 Gene #: n/a
Organism: E. coli
Device type: Actuator
Glycerol stock barcode: 0133021735
Box label: BioE44 F16