Difference between revisions of "Part:BBa K934001"

Revision as of 02:36, 26 October 2012

phaC1-A-B1 [P(3HB) synthesis]

Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes.

• The A gene encodes for the 393 amino acids protein, 3-ketothiolase (PhaA)
• The B1 gene encodes for the 246 amino acids protein, acetoacetyl-CoA reductase (PhaB)
• The C1 gene encodes for the 589 amino acids protein, PHA Synthase (PhaC)

To synthesize P(3HB) by E.coli, we transformed E.coli JM109 with the constructed phaC1-A-B1 parts on pSB1C3 (BBa_K934001). E.coli JM109 is used to synthesize P(3HB), because it tends to have a high density accumulation of P(3HB). As a negative control, we transformed E.coli JM109 with PlasI-gfp on pSB1C3.

Fig.1 synthesis mechanism of P(3HB)

The pathway and regulation of Poly[(R)-3-hydroxybutyrate] ,P(3HB) synthesis in Ralstonia eutropha H16 is shown in Fig1. Pyruvic acid is metabolized from glucose by glycolysis, and pyruvate dehydrogenase complex (PDC) transforms pyruvic acid into acetyl-CoA. At first, two molecules of acetyl-CoA are ligated to one molecule acetoacetyl-CoA by the action of 3-ketothiolase (coded in phaA). Acetoacetyl-CoA is transformed into (R)-3-hydroxybutyl-CoA by NADPH dependent acetoacetyl-CoA reductase (coded in phaB). P(3HB) is then synthesized by the polymerization of (R)-3-hydroxybutyryl-CoA by the action of PHA synthase (PhaC).

Fig.2 shows the difference between cells storing P(3HB) and those not storing P(3HB). The cells in blue rectangle area are the cells with P(3HB) synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control.

We cultured the colony in LB solution for 16hrs at 37℃, then we concentrated the solution and painted the letter by the solution on LB agar medium including 0.5μg/ml Nile red and 2% glucose at 37℃ for 36 hours. The cells with P(3HB) would be stained red by Nile red when observed under UV.

Fig2. Difference between cells storing P(3HB) and cells not storing P(3HB). Blue rectangle: with BBa_K934001 gene, P(3HB) accumulation. Green rectangle: with PlasI-gfp gene, no P(3HB) accumulation.

We successfully identified the product as 3HB, monomer of P(3HB), by Gas Chromatography/ Mass Spectrometry. To confirm 3HB by GC/ MS, 3HB is methylated because 3HB is difficult to measure. Following Fig.3, the peaks of sample are same to those of standard control of methylated 3HB. This shows E.coli synthesized P(3HB) correctly.

Fig3. Result of GC/MS

We made P(3HB) sheets. To make the sheets, we cultured E.coli JM109 in erlenmeyer flasks at 37℃ for 72h.

Fig4. P(3HB) sheet

Examine best culture condition to synthesize P(3HB)

To examine best culture condition, we tried culturing E.coli JM109 in 10 conditions for 48h. This culture is done in test tubes. Composition of LB and TB medium is shown in Fig.5.

Fig5. Composition of LB & TB

Fig6. Structure of Pantothenic acid

Pantothenic acid (PA), also called vitamin B5 is required to synthesize coenzyme A (CoA). If the glycolytic pathway has become a rate-limiting step, P(3HB) synthesis would be more efficiently by adding PA. The culture result is shown in Fig.7.

Fig7. culture result

Both in LB and TB, when we added glucose and PA-Ca, culturing at 37℃, we got maximum polymer content rate. However, the growth of E.coli in 37℃ is worse than that of in 30℃, so final polymer concentration in 37℃ and 30℃ doesn’t make significantly difference. Even if there is no glucose, E.coli synthesizes polymer (condition 10). We think TB medium has a lot of yeast extra and glycerol, so E.coli may use them as a carbon source.

In addition, according to condition 4 & 5, PA-Ca is not used as a carbon source. Since E.coli has rich carbon source in TB medium, according to condition 7 & 8 and 9 & 10, the glycolytic pathway may become a rate-limiting step, so polymer production would be increased by adding PA-Ca.

For more information, see Experience, or [http://2012.igem.org/Team:Tokyo_Tech/Projects/PHAs/index.htm#3. our work in Tokyo_Tech 2012 wiki].

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