Part:BBa_K2260000
Overview
The naturally occuring phaCAB operon in R. eutropha H16 is involved in biosynthesis of poly[(R)-3-hydroxybutyrate] (PHB) ___!!source!!____. It utilizes acetyl-coA, which is a product of the glycolysis pathway ___!!source!!____. 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 ___!!source!!____.
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 part 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 containing VFAs. 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 VFAs, OD600 of cells after growing in media for ~24 hours, then 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 because . The following figures shows the HPLC results obtained:
20 mins digestion, dilution factor 100
Figure 2. HPLC results from digestion of PHB in sulphuric acid for 20 mins.
30 mins digestion, dilution factor 100
Figure 3. HPLC results from digestion of PHB in sulphuric acid for 30 mins.
30 mins digestion, dilution factor 32
Figure 3. 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:
Figure 4. Standard curve to estimate concentration of crotonic acid in sample using Polyferm's PHB.
Table 2. PHB amount in mg calculated from amount of crotonic acid recorded in HPLC of samples digested for 20 mins (low), 30 mins (high), and arbitrary dilution factor. The area of crotonic acid recorded in HPLC, dilution factor, and conversion factor were used to calculate the amount of PHB in the three samples
Discussion of HPLC results
The HPLC results showed that a peak for crotonic acid was seen for all three conditions. This confirmed 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 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
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