Difference between revisions of "Part:BBa K2260000"

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		+	<font size="4"><b>phaCBA operon with polyhistidine tags</b></font>
	<h2>Overview</h2>		<h2>Overview</h2>

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Revision as of 04:42, 31 October 2017

phaCBA operon with polyhistidine tags

Overview

The naturally occuring phaCAB operon in R. eutropha H16 is involved in biosynthesis of poly[(R)-3-hydroxybutyrate] (PHB) (Hiroe et al., 2012). It utilizes acetyl-coA, which is a product of the glycolysis pathway (Hiroe et al., 2012). Transcription of the phaCAB operon leads to expression of the following enzymes in the order: pha synthase, acetoacetyl-CoA reductase, and 3-ketothiolase. The expression of phaA leads to expression of 3-ketothiolase that converts acetyl-coA to acetoacetyl-CoA. The acetoacetyl-CoA reductase enzyme resulting from the expression of phaB leads to conversion of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA. Finally, pha synthase leads to synthesis of PHB from (R)-3-hydroxybutyryl-CoA (Hiroe et al., 2012).

In order to utilize acetic acid present in fermented human feces, we decided to incorporate the phaCAB operon. However, literature has shown that the rearrangement of operon to phaCBA leads to higher amount of production of PHB (Hiroe et al., 2012). Thus, we obtained the operon from BBa_K1149051 and rearranged the construct from phaCAB to phaCBA and added histidine tags. The iGEM suffix is at the end of the gene construct.

PHB Weights from fermented "syn poo" supernatant

Our part was tested for production of PHB using the PHB synthesis using VFAs as feedstock protocol. There were 9 replicates for our construct in the "syn poo" supernatant and 3 replicates of the negative control (E. coli (BL21) transformed with pET29(b)+, no insert). The OD₆₀₀ of overnights (O/Ns) was measured before inoculating the media with "syn poo supernatant". PHB was extracted using the sodium hypochlorite extraction protocol. After resuspension of cells in 1x PBS for extraction the OD₆₀₀ was recorded to estimate the number of cells. The following table summarizes the data:

Table 1. Recorded OD₆₀₀ of O/Ns before inoculating the media containing "syn poo supernatant", OD₆₀₀ of cells after growing in media for ~24 hours, centrifuged, and resuspended in (1x) PBS for extraction. Initial weight of 50 mL falcon tubes and final weights of tube containing PHB was recorded in grams.

Figure 1. Tubes after sodium hypochlorite extraction of PHB from cells. Negative control on left and phaCBA on right.

HPLC of PHB digested in sulfuric acid

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. About 0.581 g of product was obtained and digested for 20 mins (Low) and 30 mins (High). The dilution factor used for these samples was 100. Lastly, a sample with a dilution factor of 32 was digested for 30 mins. The following figures shows the HPLC results obtained for sample with dilution factor of 32:

30 mins digestion, dilution factor 32

Figure 1. HPLC results from digestion of PHB in sulphuric acid for 30 mins.

A standard curve was generated using PHB from Polyferm with known concentration of PHB. The concentrations of PHB used for standard curve were 0.01 mM, 0.25 mM, 0.5 mM, and 0.75 mM. The area of crotonic acid from HPLC results was then used to calculate the concentration of PHB in sample. The standard curve generated is shown below:

The HPLC results showed that crotonic acid was present in all the three samples. However, the amount of crotonic acid in samples with dilution factor of 100 could not be quantified because the dilution factor was too high. The amount of crotonic acid detected in sample with a dilution factor of 32 was 0.0282006 mM. The dilution factor of 32 was an arbitrary value selected because the amount of PHB in product was unknown.

Discussion of HPLC results

The HPLC results confirmed that the product obtained was PHB. However, the area of crotonic acid was 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 run of the HPLC sample could give more conclusive results about the amount of PHB in our sample.

References

Hiroe A, Tsuge K, Nomura CT, Itaya M, Tsuge T. 2012. Rearrangement of gene order in the phaCAB operon leads to effective production of ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli. Appl. Environ. Microbiol. 78:3177–3184. 10.1128/AEM.07715-11.

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