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Synthesis

Overview

The overarching goal for the synthesis component of the project was to produce poly-3-hydroxybutyrate (PHB) by utilizing the nutrients present in human waste. In order to accomplish this, we:

analyzed human waste and chose organic compounds to use as feedstocks for our bacteria, and
researched and optimized efficient pathways to turn relevant components of human waste (acetyl-CoA and volatile fatty acids) into PHB

Analysis of human waste

Choice of pathways

Chassis and vector

Our gene construct was placed under an inducible promoter (lacZ, lacY). The construct was inserted into pET29(b)+ vector, which contains the promoter and kan resistant gene. The chassis used for our experiments was E. coli (BL21). The bacterium, E. coli, is known to adapt well to both aerobic and anaerobic conditions and grow quickly given an adequate carbon source. Being a popular model organism, its metabolic pathways are well-studied and, thus, provide us with an extensive array of identified molecules and structural components to manipulate. E. coli also has a better ability to express proteins (source). The operon was induced using IPTG at different concentrations.

Hiroe et al. investigated the effect that gene order of the phaCAB operon had on the PHA molecular weight and yield. They found that the molecular weight was most balanced in the strain harboring phaBCA expression plasmid. However, the highest PHA yield was found in the order phaCBA. Therefore, we decided to use this gene order to maximize pha production.

WORKS CITED

George, H.J., Watson, R.J., Harbrecht, D.F. & DeLorbe, W.J. (1987). A bacteriophage lambda cI857 cassette controls lambda pL expression vectors at physiologic temperatures. Nature Biotechnology. 5: 600-603.