Team:Caltech/BBa K338004

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PHA Synthase Composite, Part 2/2

This is half of a planned part which would contain all three PHA synthase genes required to produce polyhydroxybutyrate (PHB) in cells: phaA, phaB1, phaC1. This half contains phaB1 and phaC1: BBa_K156013 & BBa_K156014. It was designed to be ligated downstream of part BBa_K338003.

Usage and Biology

Design

When ligated downstream of BBa_K338003, the completed construct was designed to express all three PHA synthase genes required to make PHB oligomers from soybean oil. The three genes would be transcribed polycistronically on a single mRNA transcript under the IPTG-inducible control of the BBa_K215000 promoter. Naturally, each gene is preceded by a standard RBS (BBa_B0034) and the transcript finishes with a strong terminator (BBa_B0015), for a total size of about 3500bp.

Note that these three genes should only cause the production of PHB oligomers in cells, not hardened plastic. A crosslinking agent is required to link the oligomers and form the final plastic product. Over-expression of the phaC1 gene could cause some crosslinking, but this has not been experimentally verified.

Literature

SY Lee describes how a similar gene construct (pSYL105) was used to produce very large amounts of PHB, up to 80-90% of the dry cell weight, under certain conditions. Synthesis of PHB is related to the amount of acetyl-CoA available - synthesis was bolstered in the presence of complex nitrogen sources, amino acids, or oleic acid. He also mentions that PHB production was highly dependent on the particular bacterial strain used. [10]

Characterization

Although not a finished product, it was tested in DH5α cells grown in 5mM glucose for 48 hours (5mL LB culture) at 37°C. The cells were then imaged at 100x, producing the following figures:

CaltechPlastic1.jpg

CaltechPlastic2.jpg

The small inclusion bodies suggest the production of an unknown, but possibly plastic-related, substance. The pSYL105 construct mentioned above also caused the formation of large inclusion bodies in cells that produced plastic, lending credence to this possibility.

 
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