Team:Wisconsin-Madison/alkane

In the production of large chain alkanes for biofuel production, it is crucial for there to be a rapid and accurate diagnostic for comparing production rates in engineered strains of E. coli. To develop an alkane biosensor, genes from Pseudomonas putida and Alcanivorax borkumensis were isolated and constructed into a pair of plasmids which code for proteins that bind to alkanes. This induces a promoter upstream of a red fluorescent protein, which can be detected easily using various methods. Similar to our ethanol sensor, we can find a linear regression by gathering data at different concentrations of n-alkanes, allowing for the quantification of an unknown alkane concentration in a media based upon the level of fluorescence.

Experience

We attempted to use existing alkane sensor parts, K398300 (AlkS) and K398302 (PalkB), submitted by TU-Delft for this system. These genes were taken from Pseudomonas putida and we were unable to express the system sufficiently for our uses. Instead, we extracted the homologous genes from Alkanivorax borkumensis. Due to an internal PstI site, these genes cannot be submitted yet, but we have seen more robust response to alkanes.

One unforeseen issue with this system appears to be metabolic crosstalk. Specifically, it appears this system is repressed by the sugar arabinose. This is seen in figure 2, below. We intend to probe the system biochemically to determine the mechanism by which this acts, since there is no evidence of this in the literature.

Figure 1: In the presence of lactose, the PLac promoter will turn on transcription of alkS, which binds to the n-alkane. This complex then activates the PalkB promoter, which turns on transcription of our fluorescent protein.

Figure 2: Repression of fluorescence by addition of an inducer.

Learn more about: genes, plasmids.