Difference between revisions of "Part:BBa K2260001"

Revision as of 02:08, 31 October 2017 (view source) Atika ibrahim (Talk | contribs) ← Older edit		Revision as of 04:43, 31 October 2017 (view source) Rcvarga (Talk | contribs) Newer edit →
Line 2:		Line 2:
	<!--__NOTOC__-->		<!--__NOTOC__-->
	<!--<partinfo>BBa_K2260001 short</partinfo>-->		<!--<partinfo>BBa_K2260001 short</partinfo>-->
		+	<font size="4"><b>phaC1-J4 with polyhistidine tags</b></font>
	<h2> Overview</h2>		<h2> Overview</h2>

---

Revision as of 04:43, 31 October 2017

phaC1-J4 with polyhistidine tags

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 hydratase and pha 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). E. coli (BL21) was transformed with the plasmid containing the gene construct. The bacteria was used in our experiments to test the ability of the part to synthesize PHB in different conditions. The three different conditions used were pure VFAs only, fermented synthetic feces supernatant (which is referred as "syn poo" supernatant on this page), and glucose only.

PHB from fermented "syn poo" supernatant

In order to test the construct in the different conditions, overnights (O/Ns) of our construct and negative control (E. coli transformed with pET29(b)+ containing no insert) was were used. The O/Ns were grown for ~24 hours and the OD₆₀₀ was adjusted to be in the range 0.4-0.7. The table below shows the OD₆₀₀ readings taken before inoculation.

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

Three replicates were carried out for each of the conditions. Our construct and negative control O/Ns were added to the media containing carbon sources. The composition of the media in each of the 12 125 mL Erlenmeyer flasks is given below:

Table 1. Negative control and our construct in three different conditions: glucose only, pure VFAs only, and "syn poo" supernatant.

The cells were allowed to grow in the media for ~24 hours and centrifuged. Cells were then resuspended in (1x) PBS for extraction and the OD₆₀₀ was recorded before proceeding to other steps for extraction. 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 final weights of tube containing the product was weighed and recorded as follows:

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.

Figure 1. Tubes after sodium hypochlorite extraction of PHB from cells. Negative control on far right and other tubes containing our construct in the different conditions.

HPLC of PHB

Karr et. al showed that digestion of PHB in sulphuric acid leads to production of crotonic acid (1983). We analyzed the product obtained using the PHB digestion in sulphuric acid protocol. 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 3. HPLC results from digestion of PHB in sulphuric acid for 30 mins and low dilution factor.

High dilution factor

Figure 4. HPLC results from digestion of PHB in sulphuric acid for 15 mins and high dilution factor.

High dilution factor

Figure 5. 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 for all samples. This confirmed the product obtained after extraction was PHB. However, the area of crotonic acid was very low due to a number of limitations. 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. 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.

References

Karr DB, Waters JK, Emerich DW. Analysis of Poly-β-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection . Applied and Environmental Microbiology. 1983;46(6):1339-1344.

Sequence and Features