Team:Newcastle/10 August 2010
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==Gibson assembly of ''rocF'' BioBrick== | ==Gibson assembly of ''rocF'' BioBrick== | ||
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+ | =lacI and pVeg= | ||
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+ | ==Aims== | ||
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+ | We aim to extract pVeg and lacI from transformed ''E.coli'' DH5alpha (see [[Team:Newcastle/9_August_2010|09.08.2010]]) | ||
=Subtilin Immunity BioBrick= | =Subtilin Immunity BioBrick= |
Revision as of 12:23, 10 August 2010
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Contents |
Gel extraction of rocF BioBrick components
Gel Electrophoresis for Amplified Pspac_oid promoter, Double terminator and Plasmid Vector pSB1C3
Aim
The aim of the experiment is to perform gel electrophoresis for the amplified fragments of Pspac_oid promoter, double terminator and plasmid vector pSB1C3 and thus perform gel exraction of the required bands.
Materials and Protocol
Please refer to: Gel electrophoresis.
Result
The experiment is divided in two separate gels. For plasmid vector pSB1C3, we used 1% agarose gel but for Psapc_oid and double terminator fragment we used 1.5% agarose gel for a better resolution. We used 1Kb DNA ladder (Promega) for the gel containing plasmid vector while we used 100bp DNA ladder (Promega) for the gel containing Pspac_oid and double terminator fragment.
Pspac_oid pormoter | Double Terminator | Plasmid Vector pSB1C3 | |
---|---|---|---|
Size of the Fragment (in bp) | 148 approx. | 116 approx. | 2072 approx. |
Table 1: Table represents the sizes of the Pspac_oid fragment, Double terminator and plasmid vector pSB1C3 represented as bands on the gel in their respective lanes.
Discussion
We found two bands in the all lanes out of which one is of approximately of 150 bp is size and the other band is of 80 bp approximately in size.
Conclusion
Gibson assembly of rocF BioBrick
lacI and pVeg
Aims
We aim to extract pVeg and lacI from transformed E.coli DH5alpha (see 09.08.2010)
Subtilin Immunity BioBrick
Aims
Subtilin is required as a cell-signalling molecule in order to trigger calcium carbonate precipitation, as well as bacteria filament formation. We aim to produce 2 subtilin BioBricks; Production and Immunity. Because subtilin is a lantibiotic that is found naturally in Bacillus subtilis ATCC 6633, the introduction of subtilin into our Bacillus 168 cells would actually kill them. Therefore, it is essential to establish immunity for 168 strains.
Materials and Protocol
Please refer to the Phusion PCR protocol.