Part:BBa_K2260001

Overview

In order to utilize short and medium chain length volatile fatty acids (VFAs) our part consists of phaC1 (Pseudomonas aeruginosa) and phaJ4 (Pseudomonas putida). Transcription of phaJ4 leads to expression of enoyl-coA hydrates and aha synthase from phaC1. These enzymes are involved in the pathway that leads to conversion of volatile fatty acids (VFAs) such as acetic acid, propionic acid, butyric acid, etc. to poly[(R)-3-hydroxybutyrate] (PHB). The gene construct also includes histidine tags upstream of each gene and contains two ribosome binding sites (RBS).

This part was inserted into pET29(b)+ downstream a T7 promoter and lacZ. Thus, expression of phaC1J4 gene was induced using Isopropyl β-D-1-thiogalactopyranoside (IPTG). The plasmid containing the insert was then used to transform E. coli (BL21), which was used in our experiments to test the ability of the part to synthesize PHB.

PHB from fermented "syn poo" supernatant

The following table shows the different conditions the bacteria was grown:

Table 1. Three replicates for each of the condition was prepared. Negative control did not contain the phaC1J4 construct, whereas the other conditions had bacteria transformed with pET29b(+) containing the phaC1J4 insert.

The OD₆₀₀ of overnights (O/Ns) was measured before inoculating the media with bacteria. The table below shows the recorded OD₆₀₀.

Table 2. The O/Ns were grown for ~24 hours and OD₆₀₀ was adjusted and recorded in the table.

The cells were allowed to grow in media for ~24 hours and centrifuged. Cells were then resuspended in (1x) PBS for extraction and the OD₆₀₀ was measured before proceeding to other steps for extraction of PHB. The table below shows the recorded OD₆₀₀.

Table 3. The OD₆₀₀ readings of cells resuspended in (1x) PBS.

After extraction of PHB from cells using sodium hypochlorite extraction method the initial and final weights of tube containing PHB was weighed.

Table 4. Initial weight of 50 ml Falcon tubes was recorded. Final weight of tube + PHB extracted was recorded. Finally, final weight - initial weight was used to calculate the amount of PHB extracted from the cells in 50 ml cultures.

References

Sequence and Features BBa_K2260001 SequenceAndFeatures