IGEM:IMPERIAL/2008/Prototype/Wetlab/test constructs

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<html><a href=http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype><img width=50px src=http://openwetware.org/images/f/f2/Imperial_2008_Logo.png></img</a></html> Home The Project B.subtilis Chassis Wet Lab Dry Lab Notebook

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Phase 1

Characterisation of Constitutive promoter and RBS

Aim : To characterise the different combinations of constitutive promoters and RBS in terms of steady-state protein production. At the end of this testing we will have decided upon a constitutive promoter and RBS combination to use in later test constructs. The combinations of Promoter and RBS are listed below,
  1. p43-gsiB,
  2. p43-SpoVG,
  3. pVeg-gsiB,
  4. pVeg-SpoVG

Phase 2

Characterisation of inducible promoters and RBS

Varying combinations of inducible promoters and RBS will be characterized to identify an efficient combination giving high levels of protein expression. In addition, constitutive promoters previously characterised will be used to express the transcriptional regulators required for each promoter. In total Four combinations will be tried(Promoter-RBS),
  1. pHyper-spank-gsiB,
  2. pHyper-spank-SpoVG,
  3. pxyl-gsiB,
  4. pxyl-SpoVG

Characterisation of light inducible promoters and RBS

Varying combinations of light inducible promoters and RBS will be characterized to identify an efficient combination giving high levels of protein expression. In addition, constitutive promoters previously characterised will be used to express the light receptor protein YtvA required for efficient light sensing. In total Four combinations will be tried(Promoter-RBS),
  1. pctc-gsiB,
  2. pctc-SpoVG,
  3. pgsiB-gsiB,
  4. pgsiB-SpoVG

Phase 3

Characterisation of the clutch

To characterise the clutch EpsE affect on motility, EpsE is placed downstream of an inducible promoter.

Characterisation of Biomaterials

Combinations of signal peptides (SP) and Biomaterials will be placed downstream of an inducible promoter to characterise the synthesis and secretion in B.subtilis. The following combinations were used (SP-Biomaterial):
  1. SacB-Elastin
  2. SacB-Elastin
  3. LipA-EAK16-11
  4. LipA-EAK16-11


Phase 4

Light induced Expression of the clutch

Aim: To test the blue light induced expression of the clutch, EpsE and the affct of this on motility.

Light induced Expression of Biomaterials

Aim: To test the blue light induced expression and secretion of Biomaterial.



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