Team:Newcastle/13 July 2010

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=LacI BioBrick Construction=
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=Research=
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==Aims==
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We still continued to research on the urease pathway.
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To use PCR to extract lacI (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).  
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#Heidrich N, Chinali A, Gerth U and Brantl S. (2006). "The small untranslated RNA SR1 from the Bacillus subtilis genome is involved in the regulation of arginine catabolism". ''Molecular microbiology''. 62(2). 520-36.  
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#Heidrich N, Moll I and Brantl S. (2007). "In vitro analysis of the interaction between the small RNA SR1 and its primary target ahrC mRNA". ''Nucleic acids research''. 35(13). 4331-46.
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==Materials==
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Seven [[Team:Newcastle/12_July_2010|overnight cultures]]
 
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Restriction enzyme ecoR1
 
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==Protocol==
 
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*''E. coli'' DH5alpha was [[Team:Newcastle/Transformation_of_E._coli|transformed]] again with our ligation mix.
 
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*The seven overnight cultures underwent [[Team:Newcastle/Qiagen Minipreps|plasmid extraction]].
 
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*The seven plasmid extracts were then [https://2010.igem.org/TeamNewcastleRestriction_digests| restriction digested] with ecoR1 to linearise.
 
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*The seven digests were then run on a gel.
 
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==Results==
 
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This procedure was followed in order to determine whether our ''lacI'' had been ligated into the plasmid vector.
 
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[[Image:Newcastle LacI Gel 3.PNG|400px|centre]]
 
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The gel shows bands in samples 3-7 (lanes 4-8) that correspond to the size of pSB1AT3 plus the original RFP insert (4.5 Kbp) and in samples 1 and 2 (lanes 2-3) that correspond to vector only (3.5 Kbp).
 
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==Conclusion==
 
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The conclusion that can be drawn from this gel is that the lacI has not been inserted into the plasmid as we desired. However due to some confusion as to the actual size of the vector a set of double digests will be performed tomorrow to try to extract insert form the plasmid if any is present.
 
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Latest revision as of 19:38, 25 October 2010

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Research

We still continued to research on the urease pathway.

  1. Heidrich N, Chinali A, Gerth U and Brantl S. (2006). "The small untranslated RNA SR1 from the Bacillus subtilis genome is involved in the regulation of arginine catabolism". Molecular microbiology. 62(2). 520-36.
  2. Heidrich N, Moll I and Brantl S. (2007). "In vitro analysis of the interaction between the small RNA SR1 and its primary target ahrC mRNA". Nucleic acids research. 35(13). 4331-46.
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