DESIGN

Poly-3-hydroxybutyrate, P(3HB), a kind of PHAs 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)

These three enzymes can use Co-A as a substrate to produce P(3HB). IGEM team Tokyo Tech2012 designed and synthesized phaC1-A-B1gene sequence BBa_K934001, and expressed via constitutive promoter.

In order to make the production process more controllable, we added heat-sensitive promoter(BBa_K873002) and arabinose inducible promoter（BBa_I0500）to phaC1-A-B1 gene sequence （BBa_K934001）, constructing the heat and arabinose inducible parts （BBa_1891015）and （BBa_1891016）. Finally we linked the newly synthesized parts to the standard vector pSB1C3. The new plasmids can be used directly for heat shock and arabinose induced expression in E.coli.

RESULTS

1. Plasmid Proliferation

We transformed the official plasmid phaC1-A-B1（BBa_K934001）,HSP（BBa_K873002）, and pBAD（BBa_I0500）into Trans 5α for plasmid extraction. The agarose gel result showed that the plasmids are all in their correct size.

2. Vector Construction

We used Spe I and Pst I to do dual digestion in PSB1C3 which contained HSP gene and recycled gene fragments in 2117bp, used Spe I and Pst I to do dual digestion in PSB1C3 which contained pBAD gene and recycled gene fragments in 3280bp, used Xbal I and Pst I to do dual digestion in PSB1C3 and recycled gene fragments in 4208bp.

We use T4 ligase to link phaC1-A-B1 to the down stream of HSP promoter and pBAD promoter, then transform the vectors to Trans 5α for plasmid extraction. The agarose gel result showed that the plasmids are all in their correct size.

We sent the plamids with correct size to sequencing, the results showed that phaC1-A-B1 is successfully linked to the down stream of HSP promoter and pBAD promoter.

3. Induced Expression

We transformed the correct plasmids to TransB(DE3) for expression, and set different conditions for inducement as shown in table 3.

After inducement, we extracted P(3HB). The results are shown in figure 3

The result indicates that we successfully added the heat-sensitive promoter(BBa_K873002) and arabinose inducible promoter（BBa_I0500）to the up stream of P(3HB).