Team:Newcastle/Chassis
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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? | 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 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 | + | 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 | + | 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 <i>cwlD</i>, and <i>sleB</i> and <i>cwlJ</i>. | |
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. | 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 [https://2009.igem.org/Team:Newcastle/Project/Labwork/MoreProtocols#Recovery_of_cwlD_spores 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, <i>sleB</i> and <i>cwlJ</i> 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, <i>cwlD</i>. | |
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- | Using the [https://2009.igem.org/Team:Newcastle/Project/Labwork/MoreProtocols#Recovery_of_cwlD_spores treatment protocol] for the non- | + | |
We propose that we could use IPTG as a switch for germination. | We propose that we could use IPTG as a switch for germination. | ||
- | + | :''[https://2009.igem.org/Team:Newcastle/Chassis/Introduction#Bacillus_subtilis_cwlD_mutant ... Click to read more ...] | |
- | + | ==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 | |
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==Wet Lab== | ==Wet Lab== | ||
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<i>Click on the dates to go the the particular lab session.</i> | <i>Click on the dates to go the the particular lab session.</i> | ||
- | {| border="1" | + | {| class=wikitable border="1" |
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! colspan="2" | Summary of Lab Sessions for Chassis | ! colspan="2" | Summary of Lab Sessions for Chassis | ||
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| '''[https://2009.igem.org/Team:Newcastle/Labwork/8_September_2009 08/09/09] | | '''[https://2009.igem.org/Team:Newcastle/Labwork/8_September_2009 08/09/09] | ||
| Cloning of sleB | | Cloning of sleB | ||
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==BioBrick constructs== | ==BioBrick constructs== | ||
- | + | '''BBa_K174012''' | |
- | + | ''sleB'', ''Bacillus subtilis'' germination gene with RBS | |
- | + | Length: 932bp | |
- | + | [[Image:TeamNewcastleBBSleBandRBS.jpg|center|500px]] | |
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- | + | Click [http://partsregistry.org/wiki/index.php?title=Part:BBa_K174012 ''here''] for more information on this part. | |
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- | + | '''BBa_K174013''' | |
- | + | ''cwlJ'', ''Bacillus subtilis'' germination gene with RBS | |
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- | + | Length: 441bp | |
- | + | [[Image:TeamNewcastleBBCwlJandRBS.jpg|center|500px]] | |
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- | [ | + | Click [http://partsregistry.org/wiki/index.php?title=Part:BBa_K174013 ''here''] for more information on this part. |
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- | + | '''BBa_K184014''' | |
- | + | ''cwlJ'' and ''sleB'', ''Bacillus subtilis'' germination genes | |
- | + | [[Image:TeamNewcastleBBSleBandcwlJ.jpg|center|100px]] | |
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- | [ | + | Click [http://partsregistry.org/wiki/index.php?title=Part:BBa_K174014 ''here''] for more information on this part. |
- | + | ==Lab Work Strategies== | |
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- | + | [[Image:TeamNewcastleChassisFigure1.jpg|center|450px]] | |
+ | To find out more about our lab strategies, click [https://2009.igem.org/Team:Newcastle/Chassis/LabStrategies ''here'']. | ||
==<b>References:</b>== | ==<b>References:</b>== |
Latest revision as of 22:15, 21 October 2009
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.
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
Click on the dates to go the the particular lab session.
Summary of Lab Sessions for Chassis | |
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04/08/09 | Arrival of the non-germination spores. Preparation of the buffer solution required for the treatment of the spores |
07/08/09 | Preparation of the lysozyme stock solution required for treatment of the spores |
10/08/09 | Re-preparation of the buffer solution required for the treatment of the spores. Pouring of agar plates with the appropriate antibiotics. |
11/08/09 | Re-pouring the agar plates with the appropriate antibiotics |
12/08/09 | Treatment of the non-germinating cwlD spores using Method A |
13/08/09 | Results for the treatment of the cwlD spores using Method A |
17/08/09 | Repeat experiment for the treatment of the cwlD spores using Method A |
18/08/09 | Successful results for the treatment of the cwlD spores using Method A. Performed treatment for the double-knockout mutants sleB and cwlJ spores using Method A |
19/08/09 | Successful results for the treatment of the double-knockout mutants sleB and cwlJ spores using Method A |
25/08/09 | Freezing down of the treated non-germinating spores, cwlD, and sleB and cwlJ. |
02/09/09 | PCR-ing of gene sleB and cwlJ using primers previously designed and ordered. |
03/09/09 | Attempt to PCR-ing of gene sleB and cwlJ using primers previously designed and ordered again. |
04/09/09 | Redesign PCR primers |
08/09/09 | Cloning of sleB |
BioBrick constructs
BBa_K174012
sleB, Bacillus subtilis germination gene with RBS
Length: 932bp
Click [http://partsregistry.org/wiki/index.php?title=Part:BBa_K174012 here] for more information on this part.
BBa_K174013
cwlJ, Bacillus subtilis germination gene with RBS
Length: 441bp
Click [http://partsregistry.org/wiki/index.php?title=Part:BBa_K174013 here] for more information on this part.
BBa_K184014
cwlJ and sleB, Bacillus subtilis germination genes
Click [http://partsregistry.org/wiki/index.php?title=Part:BBa_K174014 here] for more information on this part.
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
News
Events
- 20 – 21 June 2009 - Europe workshop (London)
- 23 – 24 June 2009 - UK iGEM meetup (Edinburgh)
- 23 October Practice Presentation (Newcastle)
- 23 October T-shirts are ready
- 27 October Practice Presentation (Sunderland)
- 27 October Poster is ready
- 30 October – 2 November 2009 - Jamboree (Boston)
Social Net
- Newcastle iGEM Twitter
- [http://www.facebook.com/home.php#/group.php?gid=131709337641 Newcastle on Facebook]
- [http://www.youtube.com/user/newcastle2009igem Newcastle Youtube Channel]