Revision as of 01:40, 1 October 2013

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

Imperial College London 2013 Experience

Purification of P(3HB)

The Imperial College iGEM team have successfully purified P(3HB) from E. coli. (MG1655) transformed with either native phaCAB (BBa_K934001 or hybrid promoter phaCAB (BBa_K1149051). Our novel Biobrick hybrid promoter phaCAB (BBa_K1149051) produces significantly more P(3HB) than the native phaCAB operon. To find more information about the reasons for improvement, the design and methods of changing the promoter on Imperial iGEM wiki: [http://2013.igem.org/Team:Imperial_College/BioPlastic_Recycling:_PHB PHB recycling]

Comparison of P3HB production P3HB extracted from E.coli MG1655 transformed with left to right, control (empty vector), native phaCAB (BBa_K934001) and hybrid promoter phaCAB (BBa_K1149051). Each produced in 300ml cultures of LB with 3% glucose after one night growing at 37 degrees celsius.

Nile red staining of P(3HB)

O/N cultures of MG1655 transformed with either control (empty vector), native, constitutive or hybrid phaCAB constructs were spread onto LB-agar plates with 3% glucose and Nile red staining.

phaCAB P(3HB) synthesis constructs transformed into MG1655 Strains were grown on Nile red plates, which stain the PHB strongly and fluoresce in presence of PHB. On the left are MG1655 cells with an empty vector (no fluorescence; no plastic), at the bottom is the native promoter (i.e. low fluorescence, some plastic). At the top and right we have our constitutive and hybrid promoter (respectively), which both show high expression and thus fluoresce very clearly.

Conclusion: The red staining indicates the production of P(3HB). More importantly our new Biobricks hybrid promoter phaCAB BBa_K1149051 and constitutive phaCAB BBa_K1149052 produce more P(3HB) than the native phaCAB operon To find more information about the reasons for improvement, the design and methods of changing the promoter on Imperial iGEM wiki: [http://2013.igem.org/Team:Imperial_College/BioPlastic_Recycling:_PHB PHB recycling]

Initial characterisation of native phaCAB

O/N cultures of MG1655 transformed with either control or phaCAB plasmid were spread onto LB-agar plates with 3% glucose and Nile red staining.

Initial work with plastic synthesis in the native promoter. Nile red staining was used to show expression of the plastic by fluorescence imaging. Control cells with empty vector are shown on the left, while native phaCAB transformed MG1655 is on the right.

iGEM12_Tokyo_Tech

To synthesize PHB by E.coli, we transformed E.coli JM109 with the constructed phaC1-A-B1 part 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.

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.

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.

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.

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.

Using the LB solution, we painted a rose silhouette on the LB agar plate containing Nile red. (FIG3).

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.

FIG4.A E.coli JM109 cells with PHB accumulation).FIG4.B E.coli JM109 cells without PHB accumulation. Optical magnification X100).

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