Latest revision as of 15:08, 27 October 2010

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Problem

Genomic DNA extraction from Bacillus subtilis strain ATCC 6633

Aim

The aim of this experiment was to extract genomic DNA from Bacillus subtilis strain ATCC 6633. This strain has the subtilin genes, required for our Subtilin Production and Immunity parts.

Protocol

Please refer to: DNA extraction of Bacillus subtilis for materials required and protocol.

Results

Results were as expected see: https://2010.igem.org/Team:Newcastle/7_July_2010

Conclusion

See: https://2010.igem.org/Team:Newcastle/7_July_2010

Research

Today we also continued with our research on filamentous cells which will act as reinforcing fibres in the crack.

Baarle S van and Bramkamp M. (2010). "The MinCDJ system in Bacillus subtilis prevents minicell formation by promoting divisome disassembly". PloS one. 5(3). e9850.
Mo AH and Burkholder WF.(2010). "YneA, an SOS-induced inhibitor of cell division in Bacillus subtilis, is regulated posttranslationally and requires the transmembrane region for activity". Journal of bacteriology. 192(12). 3159-73.

@@ Line 4: / Line 4: @@
 ==Aim==
-The aim of this experiment was to extract genomic DNA from ''Bacillus subtilis'' strain ATCC 6633. This strain produces the subtilin genes, required for our Subtilin Production and Immunity BioBricks as well as the Quorum sensor biobrick designed by Team Newcastle 2008.
+The aim of this experiment was to extract genomic DNA from ''Bacillus subtilis'' strain ATCC 6633. This strain has the subtilin genes, required for our Subtilin Production and Immunity parts.
-==Materials and methods==
-*''ara'' forward and reverse primers
-*''sac'' forward and reverse primers
-*Pipettes
-*Universal tubes
-*Eppendorf tubes
-*Centrifuge
-*PCR (see PCR protocol from Lab training)
-*Gel electrophoresis (see Gel electrophoresis from Lab training)
-''ara'' and ''sac'' are two genes that exist in the ATCC 6633 strain. ''ara'' and ''sac'' forward and reverse primers are two tests, which will be used in PCR. At the end, Gel Electrophersis can be used to distinguish the bands. If the bands from either of ''ara'' or ''sac'' show up at the expected bps against the DNA ladder, this proves that the genomic DNA is sucessfully extracted.
 ==Protocol==
@@ Line 27: / Line 18: @@
 See: https://2010.igem.org/Team:Newcastle/7_July_2010
-=LacI BioBrick Construction=
+=Research=
+Today we also continued with our research on filamentous cells which will act as reinforcing fibres in the crack.
-==Aims==
+#Baarle S van and Bramkamp M. (2010). "The MinCDJ system in Bacillus subtilis prevents minicell formation by promoting divisome disassembly". ''PloS one''. 5(3). e9850.
-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).
+#Mo AH and Burkholder WF.(2010). "YneA, an SOS-induced inhibitor of cell division in Bacillus subtilis, is regulated posttranslationally and requires the transmembrane region for activity". ''Journal of bacteriology''. 192(12). 3159-73.
-==Materials==
-* [[Team:Newcastle/5_July_2010#LacI_BioBrick_Construction|Overnight culture]].
-* Restriction enzymes EcoR1 and Xba1.
-* PCR primers designed for ''lacI'' extraction.
-==Protocol==
-* [[Team:Newcastle/Qiagen_Minipreps|Plasmid miniprep]] from overnight cultures.
-* [[Team:Newcastle/Restriction_digests|Restriction digest]] of pSB1AT3 with EcoR1 and Xba1.
-* Amplify ''lacI'' fragment by [[Team:Newcastle/PCR|PCR]] on pMutin4.
-* [[Team:Newcastle/Restriction_digests|Restriction digest]] of amplified fragment of pMutin4 with EcoR1 and Spe1.
-==Inference==
-* The plasmids are extracted from the transformed ''E. Coli'' and digested so that they are linear molecules, ready to be run on an agarose gel. Undigested pMutin4 is used as the PCR template to amplify the lacI coding sequence.
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Team:Newcastle/6 July 2010

From 2010.igem.org