Team:Newcastle/16 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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#Klein C, Kaletta C and Entian KD. (1993). "Biosynthesis of the lantibiotic subtilin is regulated by a histidine kinase/response regulator system". ''Applied and environmental microbiology''. 59(1). 296-303.
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*[[Team:Newcastle/15_July_2010|Overnight culture]] of transformants
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#Heinzmann S, Entian KD and Stein T. (2006). "Engineering Bacillus subtilis ATCC 6633 for improved production of the lantibiotic subtilin". ''Applied microbiology and biotechnology''. 69(5). 532-6.  
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*Restriction enzymes ''Eco''R1 and ''Pst''1
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==Protocol==
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*Plasmid extraction of transformants
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*Single (''Eco''R1) and double (''Eco''R1 and ''Pst''1) restriction digest of plasmid extracts
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*Run digests on a gel
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==Results==
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[[Image:Newcastle LacI BioBrick Construction Gel.png|700px|centre]]
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'''Figure 1.''' Gel electrophoresis of the single and double digests using ''Eco''R1 and ''Pst''1.
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*'''Lane 1-7:''' Single digests of colonies 2-8 in order
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*'''Lane 8:''' 1 Kbp Molecular weight marker
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*'''Lane 9-15:''' Double digests of colonies 2-8 in order
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This gel shows that colony 2 contains only pSB1AT3 (3.5 Kbp) and that the rest contain pSB1AT3 (3.5 Kbp) with an RFP (1 Kbp)insert (total 4.5 Kbp). Also please note that the gel is in fact off angle due to being twisted and the picture not being straight on. This is as was shown in the other tests that were performed this week.
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==Conclusion==
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We can conclude from these results that our ligation attempt has failed and that in order to carry on the construction we would need to re-perform the ligation using more of the PCR product. However this will not be done for the moment for these reasons: Firstly we believe that we can use another BioBrick already in the registry that will suit our needs, this is only made possible by the looming prospect of using Gibson cloning method to construct our Bricks; Secondly there are other Bricks that our more important for our project that we need to concentrate on.
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{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Latest revision as of 20:28, 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. Klein C, Kaletta C and Entian KD. (1993). "Biosynthesis of the lantibiotic subtilin is regulated by a histidine kinase/response regulator system". Applied and environmental microbiology. 59(1). 296-303.
  2. Heinzmann S, Entian KD and Stein T. (2006). "Engineering Bacillus subtilis ATCC 6633 for improved production of the lantibiotic subtilin". Applied microbiology and biotechnology. 69(5). 532-6.
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