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 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. The OD₆₀₀ of overnights (O/Ns) was measures before inoculating the media containing VFAs. PHB was extracted using sodium hypochlorite extraction method. 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 in grams was recorded.

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

It is known from literature that digestion of PHB in sulphuric acid leads to production of crotonic acid. We analyzed PHB obtained by digesting PHB using the PHB digestion in sulphuric acid protocol. About 0.581 g of PHB was obtained and digested for 20 mins (Low) and 30 mins (High). Lastly, an arbitrary amount was digested for 30 mins. The following figures shows the HPLC results obtained from the Low, High, and Arbitrary samples:

Low

Figure 2. HPLC results from digestion of PHB in sulphuric acid for 20 mins.

High

Figure 3. HPLC results from digestion of PHB in sulphuric acid for 30 mins.

Arbitrary

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 (low, high, and arbitrary). The tables below shows the results:

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 duration.

Discussion of HPLC results

The HPLC results showed that a peak for crotonic acid was seen. This confirmed the white powder 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. Finally, more than one run of the HPLC sample would 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]