Improvement
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
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 were all in their correct size.
(the plasmid size of BBa_K934001 is 6278bp, of BBa_K873002 is 2117bp, and of BBa_I0500 is 3280bp.)
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 were all in their correct size.
(the strap size of HSP+P(3HB)+pSB1C3 is about 6325bp, and of pBAD+P(3HB)+pSB1C3 is about 7488bp.)
We sent the plamids with correct size to sequencing, the results showed that phaC1-A-B1 was successfully linked to the down stream of HSP promoter and pBAD promoter.
Induced expression
We transformed the correct plasmids to TransB(DE3) for expression, and set different conditions for inducement as shown in table 1.
Table 1 The different conditions for inducement.
| experimental group | induce conditions | ||
|---|---|---|---|
| 37℃ | 42℃ | 1 mM Arabinose | |
| pSB1C3 empty | √ | √ | √ |
| P(3HB) with natural promotor | √ | ||
| P(3HB) with HSP 1 | √ | √ | |
| P(3HB) with HSP 2 | √ | √ | |
| P(3HB) with pBAD 1 | √ | √ | |
| P(3HB) with pBAD 2 | √ | √ | |
After inducement, we extracted P(3HB). The results are shown in Fig.3.
(A,B left to right: pSB1C3 empty(37℃), pSB1C3 empty(42℃ induced), P(3HB) with natural promotor, P(3HB) with HSP 1,2(37℃), P(3HB) with HSP 1,2(42℃). C,D left to right: pSB1C3 empty(37℃), pSB1C3 empty(Arabinose induced), P(3HB) with natural promotor, P(3HB) with pBAD 1,2(37℃), P(3HB) with pBAD 1,2(Arabinose induced)
The result indicated that we successfully added the heat-sensitive promoter(BBa_K873002) and arabinose inducible promoter(BBa_I0500) to the up stream of P(3HB).
