Team:Cambridge/Bacillus

From 2008.igem.org

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=Working with ''B.subtilis''=
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'''To build more complex cellular systems, new tools and techniques are required. We are generating standardized parts, tools, and techniques for the gram-positive chassis ''B. subtillis''. Easy to handle and transform, this bacterium offers many adantages to ''E. coli', including the ability to secrete proteins and integrate DNA into the chromosome. We have designed, built, and submitted gram-positive RBSes, promoters, and shuttle vectors.As a part of this work we have confirmed single copy chromosomal insertion, demonstrated InFusion assembly, and characterized an improved GFP variant. You can find our  [[IGEM:Cambridge/2008/Turing Pattern Formation/Experiments/Bacillus subtilis transfomation| ''Bacillus'' protocols here]].'''
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Building on the work of the last year's Cambridge iGEM team, we are exploring applications of the gram-positive chassis ''B.subtillis''. Easy to handle and transform, this bacterium is much better to use than ''E.coli'' wherever protein import/export is concerned, e.g. in our signalling project. An important part of our effort is to establish standard protocols and parts to work in ''B.subtillis'', characterise control elements, and develop new vectors. You can find our  [[IGEM:Cambridge/2008/Turing Pattern Formation/Experiments/Bacillus subtilis transfomation| ''Bacillus'' protocols here]].
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=Creating new Biobrick-compatible ''B.subtilis'' vectors=
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==Improved GFP==
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[[Image:plate picture.jpg| thumb | 300px | center | Superfolder GFP along with other variants under UV light]]
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We created a codon-optimized and biobricked version of the 'Superfolder' GFP engineered by Pédelacq et al (2006). This GFP variant folds faster and glows brighter than other GFP variants. [[Team:Cambridge/Improved_GFP|Read more...]]
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==Creating new Biobrick-compatible ''B.subtilis'' vectors==
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Cambridge Vectors submitted:]
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Cambridge Vectors submitted:]
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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 [http://bioinfo.clontech.com/infusion/ 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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'''ECE166''' : High-level constitutive expression of a Green Fluorescent Protein (with the promoter Pupp)
'''ECE166''' : High-level constitutive expression of a Green Fluorescent Protein (with the promoter Pupp)
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[[Team:Cambridge/Bacillus_subtilis_transformation|Read more...]]
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[[Image: phototrans166.jpg | thumb | 300px | center | Observation with microscope (x20) : B.S. transformed with ECE166 ]]
[[Image: phototrans166.jpg | thumb | 300px | center | Observation with microscope (x20) : B.S. transformed with ECE166 ]]
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[[Image: 153Amylase.jpg | thumb | 200px | left | IA751 transformed with ECE153 : SUCCESFUL TRANSFORMATION, no zone of clearing ]]
[[Image: 153Amylase.jpg | thumb | 200px | left | IA751 transformed with ECE153 : SUCCESFUL TRANSFORMATION, no zone of clearing ]]
[[Image: 112Amylase.jpg | thumb | 200px | center | IA751 transformed with ECE112 : SUCCESFUL TRANSFORMATION for 4 colonies out of 5, no zone of clearing ]]
[[Image: 112Amylase.jpg | thumb | 200px | center | IA751 transformed with ECE112 : SUCCESFUL TRANSFORMATION for 4 colonies out of 5, no zone of clearing ]]
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[[Team:Cambridge/Bacillus_subtilis_transformation|Read more...]]
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B. subtilis Promoters Designed:
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=='''B. subtilis Promoters Designed'''==
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Cambridge Biobricks submitted:]
[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Cambridge Biobricks submitted:]
* Pupp - a strong constitutive ''Bacillus cereus'' promoter present on ''B. subtilis'' vector ECE166
* Pupp - a strong constitutive ''Bacillus cereus'' promoter present on ''B. subtilis'' vector ECE166
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** [[Team:Cambridge/Bacillus_subtilis_transformation| Successfully expressed in an episomal vector...]]
* Ppac - constitutive, present on ''B. subtilis'' vector ECE151
* Ppac - constitutive, present on ''B. subtilis'' vector ECE151
* Pspac - IPTG inducible, present on ''B. subtilis'' vector ECE151
* Pspac - IPTG inducible, present on ''B. subtilis'' vector ECE151
* Pxyl - xylose inducible, present on ''B. subtilis'' vector ECE153
* Pxyl - xylose inducible, present on ''B. subtilis'' vector ECE153
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[[Team:Cambridge/Signalling/Constructs| Read more about the constructs made...]]
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[[Team:Cambridge/Signalling/Primers| Read more about the primers used...]]
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Information about the ''B. subtilis'' vectors can be found [[Team:Cambridge/Signalling/Vectors here].
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Information about the ''B. subtilis'' vectors can be found [[Team:Cambridge/Signalling/Vectors|here]].
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===The Schematic:===
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# PCR the 4 promoters out of the corresponding ''B.subtilis'' vectors using primers containing the standard biobrick restriction sites
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# Transform TOP10 with Biobrick I732007
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# Single colony PCR the transformed TOP10 cells and miniprep the colony containing I732007
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# Digest the promoter with EcoRI and SpeI
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# Digest I732007 plasmid with EcoRI and XbaI
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# Ligate the promoter to the I732007 backbone
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# Transform TOP10 cells with the new construct which should have Ampicillin resistance
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# Single colony PCR for successfully transformed cells and miniprep for more of new construct
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# Digest the new construct with EcoRI and BamHI
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# Digest ECE112 vector with EcoRI and BamHI
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# Dirty ligation of the promoter to ECE112 vector
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# Single colony PCR for the plasmid and miniprep
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# Transform ''B. subtilis'' with the plasmid which contains amyE for integration and lacZ for beta-galactosidase assay
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# Perform beta- galatosidase assay!
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Latest revision as of 04:25, 30 October 2008


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To build more complex cellular systems, new tools and techniques are required. We are generating standardized parts, tools, and techniques for the gram-positive chassis B. subtillis. Easy to handle and transform, this bacterium offers many adantages to E. coli', including the ability to secrete proteins and integrate DNA into the chromosome. We have designed, built, and submitted gram-positive RBSes, promoters, and shuttle vectors.As a part of this work we have confirmed single copy chromosomal insertion, demonstrated InFusion assembly, and characterized an improved GFP variant. You can find our Bacillus protocols here.


Improved GFP

Superfolder GFP along with other variants under UV light

We created a codon-optimized and biobricked version of the 'Superfolder' GFP engineered by Pédelacq et al (2006). This GFP variant folds faster and glows brighter than other GFP variants. Read more...

Creating new Biobrick-compatible B.subtilis vectors

Vectors submitted: 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.

pBSint1 Vector pBSep1 Vector


Successful transformation of Bacillus

Transformation with episomal vectors

ECE166 : High-level constitutive expression of a Green Fluorescent Protein (with the promoter Pupp) Read more...


Observation with microscope (x20) : B.S. transformed with ECE166

Transformation with integration vectors

ECE153 and ECE112 : Transformation in IA751 : Amylase test

IA751 transformed with ECE153 : SUCCESFUL TRANSFORMATION, no zone of clearing
IA751 transformed with ECE112 : SUCCESFUL TRANSFORMATION for 4 colonies out of 5, no zone of clearing

Read more...


B. subtilis Promoters Designed

Biobricks submitted:

  • Pupp - a strong constitutive Bacillus cereus promoter present on B. subtilis vector ECE166
  • Ppac - constitutive, present on B. subtilis vector ECE151
  • Pspac - IPTG inducible, present on B. subtilis vector ECE151
  • Pxyl - xylose inducible, present on B. subtilis vector ECE153

Read more about the constructs made... Read more about the primers used...

Information about the B. subtilis vectors can be found here.

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