Team:Newcastle/14 July 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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=LacI BioBrick Construction=
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=Research=
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==Aims==
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We initiated our research on subtilin quorum sensing system which would provide a coordinated cell response.
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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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==Materials==
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#Kleerebezem M, Bongers R, Rutten G, Vos WM de and Kuipers OP. (2004). "Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of the spa-box in subtilin-responsive promoters". ''Peptides''. 25(9). 1415-24.
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*[[Team:Newcastle/13_July_2010|Overnight digests]]
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#Klein C, Kaletta C, Schnell N and Entian KD. (1992). "Analysis of genes involved in biosynthesis of the lantibiotic subtilin". ''Applied and environmental microbiology''. 58(1). 132-42.  
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*[[Team:Newcastle/8_July_2010|Ligation mix]]
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*Competent ''E. Coli'' DH5α
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==Protocol==
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*The overnight digest are run on a gel
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*[[Team:Newcastle/9_July_2010|Repeat]] transformation of ''E. coli'' DH5α with ligation mix
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==Results==
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The gel image was unfortunately lost, however the gel was run using all of the samples and a PCR product of known length, so as to check that the Molecular weight marker was running correctly. The results were the same as the gel run [[Team:Newcastle/13_July_2010|yesterday]], i.e. colonies 1 and 2 contain only pSB1AT3 vector (running at 3 Kbp) and the others contain pSB1AT3 with the ''rfp'' insert.
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==Conclusion==
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The conclusion that can be reached from these results is that we have not been able to get transformants with the correct plasmid/insert combination. To this end the transformation is repeated. However there is again some confusion concerning the sizes, so the samples we already have will undergo a double digest tomorrow in order to extract whatever inserts may be present.
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{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 20:15, 25 October 2010

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Research

We initiated our research on subtilin quorum sensing system which would provide a coordinated cell response.

  1. Kleerebezem M, Bongers R, Rutten G, Vos WM de and Kuipers OP. (2004). "Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of the spa-box in subtilin-responsive promoters". Peptides. 25(9). 1415-24.
  2. Klein C, Kaletta C, Schnell N and Entian KD. (1992). "Analysis of genes involved in biosynthesis of the lantibiotic subtilin". Applied and environmental microbiology. 58(1). 132-42.
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