Difference between revisions of "Part:BBa K934001"

Revision as of 11:50, 26 September 2012

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

The sequence comes from Ralstonia eutropha H16 adapted to become a standard BioBrick part.

Poly-3-hydroxybutyrate, P(3HB) is synthesized by three enzymes synthesized from phaC1，phaA，phaB1，respectively.

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 PHB 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 PHB, because it tends to have a high density accumulation of PHB. As a negative control, we transformed E.coli JM109 with PlasI-gfp on pSB1C3.

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).

Fig1.synthesis mechanism of P(3HB)

Fig2 is the photographs of E.coli colonies on Nile red positive medium taken under UV. The orange colonies in Fig2.A show that the accumulated poly-3-hydroxybutyrate, PHB in cells was stained by Nile red. This result indicates that part BBa_K934001 synthesized PHB. Fig2.B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies.

We cultured the transformant on LB agar medium plates with 0.5μg/ml Nile red and 2% glucose at 37℃ for 30 hours, then we transferred the plates to 4℃ room. After 115 hours, colonies with PHB would be stained Red by Nile red when observed under UV.

Fig2.A: E.coli JM109 colonies with BBa_K934001 gene, PHB accumulation. Fig2.B: E.coli JM109 colonies with PlasI-gfp gene, no PHB accumulation.

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

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 916
Illegal BglII site found at 1741
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 222
Illegal NgoMIV site found at 293
Illegal NgoMIV site found at 893
Illegal NgoMIV site found at 1205
Illegal NgoMIV site found at 1484
Illegal NgoMIV site found at 2136
Illegal NgoMIV site found at 2158
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 4002

@@ Line 15: / Line 15: @@
-FIG1 is the photographs of E.coli colonies on Nile red positive medium taken under UV. The orange colonies in FIG1.A show that the accumulated poly-3-hydroxybutyrate, PHB in cells was stained by Nile red. This result indicates that part BBa_K934001 synthesized PHB. FIG1.B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies.
-We cultured the transformant on LB agar medium plates with 0.5μg/ml Nile red and 2% glucose at 37℃ for 30 hours, then we transferred the plates to 4℃ room. After 115 hours, colonies with PHB would be stained Red by Nile red when observed under UV.
+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).
-[[Image:Colony2.jpg|thumb|center|500px|FIG1.A: E.coli JM109 colonies with BBa_K934001 gene, PHB accumulation.
-FIG1.B: E.coli JM109 colonies with PlasI-gfp gene, no PHB accumulation.]]
-FIG2 shows the difference between cells storing PHB and those not storing PHB. The cells in blue rectangle area are the cells with PHB synthesis gene and the cells in green rectangle area are the cells with PlasI-gfp gene as a negative control.
+[[Image:metabolism.png|thumb|right|200px|Fig1.synthesis mechanism of P(3HB)]]
+Fig2 is the photographs of E.coli colonies on Nile red positive medium taken under UV. The orange colonies in Fig2.A show that the accumulated poly-3-hydroxybutyrate, PHB in cells was stained by Nile red. This result indicates that part BBa_K934001 synthesized PHB. Fig2.B is the photograph of negative control cells. In this figure we observed that there were no remarkable colored colonies.
-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.
+We cultured the transformant on LB agar medium plates with 0.5μg/ml Nile red and 2% glucose at 37℃ for 30 hours, then we transferred the plates to 4℃ room. After 115 hours, colonies with PHB would be stained Red by Nile red when observed under UV.
+[[Image:Colony2.jpg|thumb|center|500px|Fig2.A: E.coli JM109 colonies with BBa_K934001 gene, PHB accumulation.
-[[Image:PHB+.jpg|thumb|center|300px|FIG2 Difference between cells storing PHB and cells not storing PHB. Blue rectangle: with BBa_K934001 gene, PHB accumulation. Green rectangle: with PlasI-gfp gene, no PHB accumulation.]]
+Fig2.B: E.coli JM109 colonies with PlasI-gfp gene, no PHB accumulation.]]
-Using the LB solution, we painted a rose silhouette on the LB agar plate containing Nile red. (FIG3).
-[[Image:rose1.jpg|thumb|center|600px|FIG.3 Rose silhouette on the LB agar plate containing Nile red.]]
-FIG4.A is the photograph of dried E.coli (with phaC1-A-B1 gene) cells dyed with Nile blue A solution taken by fluorescence microscope. The fluorescent areas in FIG4.a are the accumulated PHB in the cells was. This result also indicates that part BBa_K934001 synthesized PHB. In the photograph of negative control (FIG4.B), no remarkable fluorescent area was observed.
-To take this photo we did shaking culture at 37 ° C for 96 hours. Then, we froze dry the cells and stained them by Nile blue A.
-[[Image:flu2.jpg|thumb|center|600px|FIG4.A E.coli JM109 cells with PHB accumulation).FIG4.B E.coli JM109 cells without PHB accumulation. Optical magnification X100).]]