PHA production detail

Other Teams

iGEM10_Caltech

Part number	Description	States	experience
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K338003 BBa_K338003]	PHA Synthase Composite, Part 1/2	planning
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K338004 BBa_K338004]	PHA Synthase Composite, Part 1/2	Available

They couldn’t prove that their engineered bacteria produced PHB according to the team wiki.

iGEM10_INSA-Lyon

Part number	Description	States	experience
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K342001 BBa_K342001]	Pha C (poly-β-hydroxybutyrate polymerase)	available

iGEM09_Duke

Part number	Description	States	experience
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K282000 BBa_K282000]	phaAB	available

State iGEM08_Utah State

Part number	Description	States	experience
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K089001 BBa_K089001]	phaA gene	planning
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K089002 BBa_K089002]	phaB gene	planning
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K089003 BBa_K089003]	phaC gene	planning

iGEM08_tsinghua

Part number	Description	States	experience
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K282000 BBa_K282000]	phaAB	available

iGEM08_Hawaii

Part number	Description	States	experience
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125801 BBa_K125801]	RBS-phaA	available
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125802 BBa_K125802]	RBS-phaB	Planning
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125803 BBa_K125803]	RBS-phaC	Available
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125804 BBa_K125804]	RBS-phaE	Available
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125501 BBa_K125501]	phaA BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)	Available
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125502 BBa_K125502]	phaB BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)	Available
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125503 BBa_K125503]	phaC BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)	Available
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K125504 BBa_K125504]	phaE BioPlastic polyhydroxybutyrate synthesis pathway (origin PCC6803 slr1994)	Available

iGEM08_Virginia

Part number	Description	States	experience
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156031 BBa_K156031]	RBS + phaA + double terminator	available
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156033 BBa_K156033]	RBS + phaB1 + double terminator	available
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156034 BBa_K156034]	RBS + phaC1 + double terminator	available
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156012 BBa_K156012]	phaA (acetyl-CoA acetyltransferase)	available
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156013 BBa_K156013]	phaB1 (acetyacetyl-CoA reductase)	available
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156014 BBa_K156014]	phaC1 (Poly(3-hydroxybutyrate) polymerase)	available
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156021 BBa_K156021]	Promoter + RBS + phaA + double terminator	available
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156018 BBa_K156018]	Promoter + RBS + phaA	planning
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156019 BBa_K156019]	Promoter + RBS + phaB1	planning
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156020 BBa_K156020]	Promoter + RBS + phaC1	planning
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156022 BBa_K156022]	Promoter + RBS + phaB1 + double terminator	planning
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K156023 BBa_K156023]	Promoter + RBS + phaC1 + double terminator	planning

PHB production by E.coli & Confirmation of PHB

text (ⅰ)

We observed the accumulation of PHB in the E.coli colonies on Nile red positive medium under UV .Nile red has been widely used to stain colonies and distinguish between PHA-accumulating and non-accumulating colonies. Nile red produces a strong orange fluorescence (emission maximum,598nm) with an excitation wavelength of 543nm (maximum) upon binding to PHA-granules in cells of R.eutropha (Degelau et al.1995) H16. Nile red in the agar medium doesn’t affect the growth of the cells, and the occurrence of PHAs in the colonies can be directly monitored. This method is quite sensitive and results in fluorescent colonies of PHA-positive strains. So 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. This showed that the transformant had stored PHB.FIG1 is the photograph of E.coli(with phaC1AB1 gene) colonies on Nile Red positive medium taken under UV. The fluorescent area in the figure showed the accumulated PHBs stained by Nile red in cells. This indicated that part BBa_K934001 functioned correctly.FIG2 is the photograph of negative control cells. In this figure we observed that there was no remarkable fluorescent area.

(ⅱ)

To confirm the accumulation condition of PHB in E.coli with a microscope, we stained the PHB with Nile blue A reagent. Nile blue A is also used to detect the existence of PHBs and has no toxicity to the cells. We carried out the observation after the procedures stated below. First，We cultured a small amount of the transformant (3ml) in LB medium for 17 hrs. Then，we added the cultured trandformant(1%) into LB medium(with 2% glucose) and shaking cultured it under aerobic condition with an air permeable lid for 96 hrs. After the cultivation we recovered the bacteria with PHA accumulation by centrifugation. We were able to get the dried cells after the procedure of lyophilization. Before taking the photographs，we stained the dried cells with 1% Nile blue A solution for 8 minutes and washed the extra Nile blue with 8% acetic acid solution afterwards. We then took photographs of the sample under fluorescence microscope. FIG1 is the photograph of E.coli(with phaC1AB1 gene) colonies on Nile Red positive medium taken under UV. The fluorescent area in the figure showed the accumulated PHBs stained by Nile red in cells. This indicated that part BBa_K934001 functioned correctly.FIG2 is the photograph of negative control cells. In this figure we observed that there was no remarkable fluorescent area.