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

1.1 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 1.PNG

Phase 2

2.1 Characterisation of inducible promoters and RBS

Aim: To characterise the different combinations of inducible promoters and RBS in terms of response time, transfer function and maximum steady-state level of protein. Each of these constructs required the use of the constitutive promoter-RBS to express the transcriptional regulators. By the end of this testing, our extensive characterisation of the different combinations would allow us to make a decision on which inducible promoter to use for Phase 3.
  1. pHyper-spank-gsiB,
  2. pHyper-spank-SpoVG,
  3. pxyl-gsiB,
  4. pxyl-SpoVG
Phase 2-linduced.PNG
2.2 Characterisation of light inducible promoters and RBS

Aim: 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, 4 combinations will be tried (Promoter-RBS),
  1. pctc-gsiB,
  2. pctc-SpoVG,
  3. pgsiB-gsiB,
  4. pgsiB-SpoVG
Phase 2-light induced.PNG

Phase 3

3.1 Characterisation of the clutch

To characterise the effect of expression of the clutch, EpsE, on motility, EpsE is placed downstream of an inducible promoter.
Phase 2-linduced22.PNG

3.2 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. LipA-Elastin
  3. SacB-EAK16-11
  4. LipA-EAK16-11
Phase 2-biomaterials.PNG


Phase 4

4.1 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.
Phase 4-clutch.PNG
4.2 Light induced Expression of Biomaterials

Aim: To test the blue light induced expression and secretion of Biomaterial.
Phase 4-clutch.PNG



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