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We still continued to research on the urease pathway.

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.
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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-=LacI BioBrick Construction=
+=Research=
-==Aims==
+We still continued to research on the urease pathway.
-*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).
+#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.
+#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.
-==Materials==
-*The seven [[Team:Newcastle/12_July_2010|overnight cultures]]
-*Restriction enzyme ''Eco''R1
-==Protocol==
-*''E. coli'' DH5α was [[Team:Newcastle/Transformation_of_E._coli|transformed]] again with our ligation mix.
-*The seven overnight cultures underwent [[Team:Newcastle/Qiagen Minipreps|plasmid extraction]].
-*The seven plasmid extracts were then [https://2010.igem.org/TeamNewcastleRestriction_digests| restriction digested] with ''Eco''R1 to linearise.
-*The seven digests were then run on a gel.
-==Results==
-[[Image:Newcastle LacI Gel 3.PNG|400px|centre]]
-'''Figure 1''': Gel electrophoresis of the seven plasmid extracts which have been digested with ''Eco''R1.
-*'''Lane 1''': 1 Kbp Molecular weight marker
-*'''Lane 2-8''': Colonies 1-7 in order
-*'''Lane 9''': 1 Kbp Molecular weight marker
-==Discussion==
-This procedure was followed in order to determine whether our ''lacI'' had been ligated into the pSB1AT3 in the way we desired (i.e. in front of RFP).
-The gel shows bands for colonies 1 and 2, that indicates that they contain vector only (size 3-3.5 Kbp) and for 3-7 that indicate that they contain vector plus RFP (size 4.5 Kbp), as with the original plasmid stock.
-==Conclusion==
-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 the feint second band we are unsure as to whether plasmid has fully digested, to that end we will leave the digest running overnight and run the samples on a gel tomorrow.
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Team:Newcastle/13 July 2010

From 2010.igem.org

Latest revision as of 19:38, 25 October 2010

Research