Team:Newcastle/26 August 2010

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=First transformation of ''Bacillius subtilis 168'' with Prrnb-GFP containing ''YneA''=
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=Screening ''Bacillius subtilis'' transformants=
==Aim==
==Aim==
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The aim of the experiment is to perform insert the plasmid Prrnb-GFP containing YneA which have been ligated eariler into the chromosome of 'Bacillus subtilis 168'. 'B. subtilis' containing the integrated vector will be resistance to both the antibiotic chloramphenicol and streptomycin, therefore those that have successful integrated will be selected with agar plates that contain these antibiotic. The second step will be to identify those colones that have the plasmid integrated at the corerct position in the chromosome, which is the amylase locus. Thus those that have integrated 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 identify those colones that have the plasmid integrated at the correct position in the chromosome, which is the ''amyE'' locus. Those that have integrated at the correct position will not be able to break down starch, which can be tested by exposing the colonies on the starch plates to iodine.
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==Materials and Protocol==
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Please refer to: [[Team:Newcastle/Transformation of B. subtilis| Transformation of ''Bacillus subtilis'']]
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Note: Overnight culture of 'B. subtilis 168' in MM competence medium was done the day before and the iodine test was performed the day after.
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===Results===  
===Results===  
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Unfortunately our colonies had halos. Although the insert has integrated, the colonies have antibiotic restistance, the insert has not integrated at the ''amyE'' locus.  
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Some of our colonies did not have halos, therefore the transformation and integration was successful for these colonies.
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Below you can see cells from these positive colonies under the microscope.
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However when we checked the colonies under a microscope the cells were filamentous due to integration elsewhere on the ''Bacillus subtilis ''168 chromosome perhaps at the native ''yne''A locus.
 
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Latest revision as of 01:15, 28 October 2010

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Screening Bacillius subtilis transformants

Aim

The aim of the experiment is to identify those colones that have the plasmid integrated at the correct position in the chromosome, which is the amyE locus. Those that have integrated at the correct position will not be able to break down starch, which can be tested by exposing the colonies on the starch plates to iodine.

Results

Some of our colonies did not have halos, therefore the transformation and integration was successful for these colonies.

Below you can see cells from these positive colonies under the microscope.

Starchplate.jpg
Starchplate2.jpg
Filamentous cells
Filamentous cells showing GFP signal
Normal Bacillus subtilis 168



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