Team:Newcastle/2 September 2010

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==Aim==
==Aim==
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The aim of the experiment is to perform insert the plasmid pGFP-rrnB containing ''yneA'' which have been ligated eariler into the chromosome of ''Bacillus subtilis'' 168. ''B. subtilis'' containing the intergated vector will be resistance to both the antibiotics chloramphenicol and streptomycin, therefore those that have successful intergated will be selected with agar plates that contain both the antibiotics. The second step will be to identify those colones that have the plasmid intergated at the corerct position in the chromosome, which is the amylase gene locus. Thus those that have intergrated at the wrong position will not be able to break down starch, which can be tested with the iodine test.
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The aim of the experiment is to transform ''Bacillus subtilis'' 168 with the plasmid pGFP-rrnB containing our filamentous cell part. After transformation the plasmid will integrate into the chromosome at ''amyE''. This integration removes the ability to break down starch, allowing us to select for our desired construct using the iodine test.
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[[Image:filamentous_in_pgfprrnb.jpg|800px]]
[[Image:filamentous_in_pgfprrnb.jpg|800px]]

Revision as of 14:13, 27 October 2010

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Contents

pH changing for HEPES

We prepared 100mM of HEPES (it is a dibasic compound) at pH 7.0 and sent it for autoclaving. N.B. During autoclaving we loose some volume of the solution.

Transformation of Bacillius subtilis 168 with pGFP-rrnB containing yneA

Aim

The aim of the experiment is to transform Bacillus subtilis 168 with the plasmid pGFP-rrnB containing our filamentous cell part. After transformation the plasmid will integrate into the chromosome at amyE. This integration removes the ability to break down starch, allowing us to select for our desired construct using the iodine test.


Filamentous in pgfprrnb.jpg

Materials and Protocol

Please refer to: Transformation of Bacillus subtilis Note: Overnigth culture of B. subtilis 168 in MM competence medium was done the day before and the iodine test was performed the day after transformation.

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