Team:Newcastle/Chassis

=Chassis=

Introduction
The main aim of our project is to sequester cadmium in the environment into the spores of our engineered B. subtilis, but what happens after the cadmium has been sequestered?

Do we attempt to retrieve the sequestered cadmium? Or do we simply leave the sequestered cadmium in the spores of our engineered B. subtilis?

For our project, we have chosen the latter. We will not be attempting to retrieve the sequestered cadmium. However, then comes the question: would there not be chances of the cadmium entering the environment again?

Our solution to this question is to disable germination of the spores. If spores cannot germinate retrieval of the sequestered cadmium is unnecessary, since the spores can persist intact for thousands of years.

We were fortunate enough that Prof. Anne Moir from Sheffield University kindly sent us two non-germinating strains, with inactivated genes, namely cwlD, and sleB and cwlJ.

While we would like to disable germination for the spores that contain sequestered cadmium, not all the cells would have sequestered cadmium, and it is also essential that we still have some cells germinating, so that our population of bacteria can continue to live and grow, reaching a balance, and not simply deplete totally.

Using the treatment protocol for the non-germinating spores from Prof. Anne Moir, we performed lab experiments for the two non-germinating spores, and concluded that the double-knockout mutant, sleB and cwlJ would be best for our project as it had more colonies growing after treatment, and fewer colonies growing without treatment, as compared to the single knock-out mutant, cwlD.

We propose that we could use IPTG as a switch for germination.


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Novelty in this sub-project
In this sub-project, we are disabling germination, using non-germinating spores with the inactivated genes, sleB and cwlJ. In order to control germination, we intend to use IPTG as a switch, via the promoter, pSpac

Wet Lab
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BioBrick constructs
BBa_K174012

sleB, Bacillus subtilis germination gene with RBS

Length: 932bp



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BBa_K174013

cwlJ, Bacillus subtilis germination gene with RBS

Length: 441bp



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BBa_K184014

cwlJ and sleB, Bacillus subtilis germination genes



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Lab Work Strategies


To find out more about our lab strategies, click here.

References:
[1] Popham, D., Helin, J., Costello, C & Setlow, P. (1996). Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore rehydration or heat resistance. ''Proc. Natl. Acad. Sci.'' 93; 15403-15410

[2] Chirakkal, H., O'Rourke, M., Atrih, A., Foster, S. J., Moir, A. (2002.) Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination. Microbiology 148; 2383-2392