Team:Imperial College/PHB production

From 2013.igem.org

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O/N cutures of MG1655 transformed with either control or phaCAB plasmid were spread onto LB-agar plates with 3% glucose and Nile red staining.
O/N cutures of MG1655 transformed with either control or phaCAB plasmid were spread onto LB-agar plates with 3% glucose and Nile red staining.
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|[[File:Nilered.JPG|thumbnail|right|400px|<b>Initial work with plastic synthesis in the native promoter.</b> 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. This was imaged by Iain.]]  
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|[[File:Nilered.JPG|thumbnail|right|400px|<b>Initial work with plastic synthesis in the native promoter.</b> 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.]]  
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|[[File:27-9-13phaCABall.jpg|thumbnail|right|400px|<b>MG1655 constructs synthesising plastic.</b> 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. This was imaged by Iain.]]
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|[[File:27-9-13phaCABall.jpg|thumbnail|right|400px|<b>MG1655 constructs synthesising plastic.</b> 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.]]
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Revision as of 20:07, 29 September 2013

PHB production

During our project we successfully synthesised the bioplastic P(3HB).

Nile red staining

O/N cutures 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.
MG1655 constructs synthesising plastic. 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 [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1149051 hybrid promoter phaCAB BBa_K1149051] and [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1149052 constitutive phaCAB BBa_K1149052] produce more P(3HB) than the native phaCAB operon


Purification of P3HB

P(3HB) purified from phaCAB transformed MG1655 that were grown in LB with 3% glucose
P(3HB) purified from phaCAB transformed MG1655 that were grown in LB with 3% glucose
P(3HB) purified from phaCAB transformed MG1655 that were grown in M9M mixed waste.

Our Sponsors

TueSponsorsEppendorf.png 125px Invitrogen.jpg Geneart.jpg CSynBI.JPG

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