Team:LMU-Munich/Bacillus BioBricks/Sporovector

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=='''Sporo'''vector==
=='''Sporo'''vector==
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<p align="justify">
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Our '''Sporo'''vector is specially designed to give you the opportunity to easily create Sporobeads with any proteins you can think of. It is available as BioBrick in pSB1C3 and can be cloned in any of our empty ''B. subtilis'' vectors. We also offer it “precloned” in pSB<sub>Bs</sub>4S which in this version lacks ''NgoM''IV restriction sites.
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Our '''Sporo'''vector is specially designed to give you the opportunity to easily create Sporobeads with any protein you can think of. The part between the ''EcoR''I and ''Pst''I is available as BioBrick in pSB1C3 and can be cloned in any of our empty ''B. subtilis'' vectors. We also offer it “precloned” in pSB<sub>''Bs''</sub>4S, which in this version lacks the ''NgoM''IV restriction sites.
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[[File:Sporovectorcloning.png|600px]]
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</p>
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The '''Sporo'''vector was created by ligation of three cut PCR-products. It is designed to allow N-terminal fusion of genes in Freiburg standard ([http://dspace.mit.edu/bitstream/handle/1721.1/45140/BBF_RFC%2025.pdf?sequence=1 RCF25]). Therefore, the gene to be inserted must be cut with ''Xba''I and ''Age''I and the vector must be digested with ''Xba''I and ''NgoM''IV. By this digest, the RFP cassette will be removed from this vector to allow easy selection for the integration of the new insert. After ligation, there is a scar of six nucleotides between both genes but no restriction site left.  
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[[File:Sporovectorcloning.png|600px|center]]
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The gene fusion is controlled by the P<sub>''cot''YZ</sub>-promoter which was the strongest one in our promoter evaluations. The C-terminal gene part is ''cge''A, one of the two known spore crust proteins.
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<p align="justify">
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The '''Sporo'''vector was created by ligation of three cut PCR-products with special overhangs (P<sub>''cotYZ''</sub>, ''mRFP'' and ''cgeA''-B0014). It is designed to allow N-terminal fusions of genes in Freiburg standard ([http://dspace.mit.edu/bitstream/handle/1721.1/45140/BBF_RFC%2025.pdf?sequence=1 RCF25]). Therefore, the gene to be inserted must be cut with ''Xba''I and ''Age''I and the vector must be digested with ''Xba''I and ''NgoM''IV. By this digest, the RFP cassette is removed from this vector to allow easy selection for the integration of the new insert. After ligation, there is a scar of six nucleotides between both genes but no restriction site left. </p>
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<p align="justify">
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Expression of the gene fusion is controlled by the P<sub>''cot''YZ</sub>-promoter which was the strongest one in our promoter evaluations. The C-terminal gene part is ''cge''A, one of the two known spore crust proteins.
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</p>
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<p align="justify">
The vector is cloned but has not been tested with an insert, yet.
The vector is cloned but has not been tested with an insert, yet.
We are working on further '''Sporo'''vectors to offer also fusion with ''cot''Z as well as C-terminal fusions.
We are working on further '''Sporo'''vectors to offer also fusion with ''cot''Z as well as C-terminal fusions.
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</p>
Back to our [[Team:LMU-Munich/Bacillus_BioBricks |'''''Bacillus''B'''io'''B'''rick'''B'''ox]]
Back to our [[Team:LMU-Munich/Bacillus_BioBricks |'''''Bacillus''B'''io'''B'''rick'''B'''ox]]

Latest revision as of 00:23, 27 September 2012

iGEM Ludwig-Maximilians-Universität München Beadzillus

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