Latest revision as of 04:25, 30 October 2008

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Bacillus Overview Transformation RBS & Promoters Primers InFusion Superfolder GFP Lab Notebook

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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

[http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Cambridge Vectors submitted:] 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.

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

B. subtilis Promoters Designed

[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
- Successfully expressed in an episomal vector...
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

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

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@@ Line 16: / Line 16: @@
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-=Working with ''B.subtilis''=
+'''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]].'''
-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]].
-=Creating new Biobrick-compatible ''B.subtilis'' vectors=
+==Improved GFP==
+[[Image:plate picture.jpg| thumb | 300px | center | 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. [[Team:Cambridge/Improved_GFP|Read more...]]
+==Creating new Biobrick-compatible ''B.subtilis'' vectors==
 [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2008&group=Cambridge 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''.
+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''.
 {| class="wikitable" border="0" align="center"
@@ Line 49: / Line 53: @@
-=='''B. subtilis Promoters Designed'''===
+=='''B. subtilis Promoters Designed'''==
 [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
@@ Line 59: / Line 63: @@
 [[Team:Cambridge/Signalling/Primers| Read more about the primers used...]]
-Information about the ''B. subtilis'' vectors can be found [[Team:Cambridge/Signalling/Vectors here].
+Information about the ''B. subtilis'' vectors can be found [[Team:Cambridge/Signalling/Vectors|here]].
 <html>

Team:Cambridge/Bacillus

From 2008.igem.org