IGEM:Cambridge/2008/Notebook/Bacillus

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=Creating new Biobrick-compatible ''B.subtilis'' vectors=
=Creating new Biobrick-compatible ''B.subtilis'' vectors=
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We are using the In-Fusion™ PCR method from ClonTech to create new Biobrick-compatible integrative and episomal vectors. These vectors will allow us to insert Biobricks into the ''Bacillus subtilis'' genome at the AmyE locus and as a 3-5 copy plasmid that will replicate autonomously in ''Bacillus''.
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We are using the InFusion PCR method from ClonTech to create new Biobrick-compatible integrative and episomal vectors. These vectors will  
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{| class="wikitable" border="0" align="center"
 
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| [[image:PBSINT1_final.png|300px|pBSint1 Vector]]
 
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| [[image:PBSEP1_final.png|300px|pBSep1 Vector]]
 
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|-
 
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|}
 
=Testing ''B.subtilis'' promoters and RBSs=
=Testing ''B.subtilis'' promoters and RBSs=
We are seeking to test the expression strength and response to inducers in several promoters in ''B.subtilis'', in combination with different Bacillus-specific ribosome binding sites.  
We are seeking to test the expression strength and response to inducers in several promoters in ''B.subtilis'', in combination with different Bacillus-specific ribosome binding sites.  
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==B. subtilis Promoter Testing with Beta-galactosidase Assay==
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=B. subtilis Promoter Testing with Beta-galactosidase Assay=
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We are aiming to characterize four different Bacillus subtilis promoters using the beta-galactosidase assay. (Protocol for beta-galactosidase assay as described [http://openwetware.org/wiki/Beta-Galactosidase_Assay_(A_better_Miller) here]
We are aiming to characterize four different Bacillus subtilis promoters using the beta-galactosidase assay. (Protocol for beta-galactosidase assay as described [http://openwetware.org/wiki/Beta-Galactosidase_Assay_(A_better_Miller) here]
B. subtilis Promoters to be Tested:
B. subtilis Promoters to be Tested:
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* Pupp  
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* Pupp - constitutive, found ''Bacillus cereus''.
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* Ppac  
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* Ppac - constitutive
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* Pspac  
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* Pspac - lactose inducible
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* Pxyl  
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* Pxyl - xylose inducible
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Revision as of 09:12, 5 September 2008


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Creating new Biobrick-compatible B.subtilis vectors

We are using the InFusion PCR method from ClonTech to create new Biobrick-compatible integrative and episomal vectors. These vectors will

Testing B.subtilis promoters and RBSs

We are seeking to test the expression strength and response to inducers in several promoters in B.subtilis, in combination with different Bacillus-specific ribosome binding sites.

B. subtilis Promoter Testing with Beta-galactosidase Assay

We are aiming to characterize four different Bacillus subtilis promoters using the beta-galactosidase assay. (Protocol for beta-galactosidase assay as described here

B. subtilis Promoters to be Tested:

  • Pupp - constitutive, found Bacillus cereus.
  • Ppac - constitutive
  • Pspac - lactose inducible
  • Pxyl - xylose inducible



Links

B. Subtilis Transformation Protcol - Electroporation

Bongers et al. … of a Subtilin-Regulated Expression System in Bacillus subtilis: Strict Control of Gene Expression …. Applied and Environmental Microbiology (2005)

  • SURE: handy expression system for B. subtilis
  • What about feedback/regulation effects of subtilin on the cell

Expression and characterization of aiiA gene from Bacillus subtilis BS-1.

  • (Some strains of) Bacillus subtilis produces a gene called aiiA that degrades AHL molecules from gram-negative bacteria
  • this is biobricked

Links to Cambridge 2007 Wiki



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