Difference between revisions of "Part:BBa K2260001"

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Revision as of 07:18, 30 October 2017

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.

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.

!!! insert image !!!

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.

HPLC of PHB

It is known from literature that digestion of PHB in sulphuric acid leads to production of crotonic acid. We analyzed the PHB obtained from extraction carried out on cells containing phaC1J4 genes by digesting the PHB in sulfuric acid. The protocol used for PHB digestion is given here. About ___!!!___ g of PHB was digested for 15 mins and 30 mins. For each of the durations low and high dilution factors were used. The following figure shows the HPLC results obtained from the samples:

Low dilution factor

Figure 2. HPLC results from digestion of PHB in sulphuric acid for 15 mins and low dilution factor.

Low dilution factor

Figure 2. HPLC results from digestion of PHB in sulphuric acid for 30 mins and low dilution factor.

High dilution factor

Figure 2. HPLC results from digestion of PHB in sulphuric acid for 15 mins and high dilution factor.

High dilution factor

Figure 2. HPLC results from digestion of PHB in sulphuric acid for 30 mins and high dilution factor.

Discussion of HPLC results

The HPLC results showed that a peak for crotonic acid was seen. This confirmed the white powder obtained after extraction was PHB. However, the area of crotonic acid was very low, which could be due to a number of reasons. The samples could not be digested in sulfuric acid for longer period of time because crotonic acid starts degrading. Thus, the amount of crotonic acid recorded may be lower than the actual amount produced. Furthermore, the conversion factor was low (i.e. below 1), which may lead to lower amount of crotonic acid calculated. Finally, more than one try of the HPLC for the samples would be effective in obtaining more conclusive results about the amount of PHB in sample.

Nile red suspension for confirmation of PHB

References

Sequence and Features