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 simply leave the sequestered cadmium in the spores of our engineered ''B. subtilis''?
+
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 of, would there not be chances of the cadmium entering the environment again?
+
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 would be to disable germination of the spores, thus retrieval of the sequestered cadmium becomes unnecessary, as the spores can persist intact for thousands of years.
+
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.
-
In order to disable germination of the spores, we would require non-germinating spores, and we were fortunate enough that Prof. Anne Moir from Sheffield University kindly sent us two non-germination spores, namely cwlD, and sleB and cwlJ.
+
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>.
-
Using the [https://2009.igem.org/Team:Newcastle/Project/Labwork/MoreProtocols#Recovery_of_cwlD_spores protocol] given to us, we performed lab experiments for the two non-germination spores, and concluded that the double-knockout mutant, sleB and cwlJ would be more ideal for our project as it had more colonies growing after treatment, and less colonies growing without treatment, as compared to the single knock-out mutant, cwlD.
+
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>.
 +
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==
==Novelty in this sub-project==
-
==Modelling==
+
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==
 +
 
 +
<i>Click on the dates to go the the particular lab session.</i>
 +
 
 +
{| class=wikitable border="1"
 +
|-
 +
! colspan="2" | Summary of Lab Sessions for Chassis
 +
|-
 +
| <center>'''Date'''</center>
 +
| <center>'''Description'''</center>         
 +
|-
 +
| '''[https://2009.igem.org/Team:Newcastle/Labwork/4_August_2009#Sporulation_Tuning.2FChassis_Team 04/08/09]'''
 +
| Arrival of the non-germination spores. Preparation of the buffer solution required for the treatment of the spores
 +
|-
 +
| '''[https://2009.igem.org/Team:Newcastle/Labwork/7_August_2009#Sporulation_Tuning.2F_Chassis_Team 07/08/09]'''
 +
| Preparation of the lysozyme stock solution required for treatment of the spores
 +
|-
 +
| '''[https://2009.igem.org/Team:Newcastle/Labwork/10_August_2009#Sporulation_Tuning.2F_Chassis_team 10/08/09]'''
 +
| Re-preparation of the buffer solution required for the treatment of the spores. Pouring of agar plates with the appropriate antibiotics.
 +
|-
 +
| '''[https://2009.igem.org/Team:Newcastle/Labwork/11_August_2009#Sporulation_Tuning.2F_Chassis_team 11/08/09]'''
 +
| Re-pouring the agar plates with the appropriate antibiotics
 +
|-
 +
| '''[https://2009.igem.org/Team:Newcastle/Labwork/12_August_2009#Sporulation_Tuning.2FChassis_Team 12/08/09]'''
 +
| Treatment of the non-germinating <i>cwlD</i> spores using Method A
 +
|-
 +
| '''[https://2009.igem.org/Team:Newcastle/Labwork/13_August_2009#Sporulation_Tuning.2FChassis_Team 13/08/09]'''
 +
| Results for the treatment of the <i>cwlD</i> spores using Method A
 +
|-
 +
| '''[https://2009.igem.org/Team:Newcastle/Labwork/17_August_2009#Sporulation_Tuning.2F_Chassis_Team 17/08/09]'''
 +
| Repeat experiment for the treatment of the <i>cwlD</i> spores using Method A
 +
|-
 +
| '''[https://2009.igem.org/Team:Newcastle/Labwork/18_August_2009#Sporulation_Tuning.2F_Chassis_Team 18/08/09]'''
 +
| Successful results for the treatment of the cwlD spores using Method A. Performed treatment for the double-knockout mutants <i>sleB</i> and <i>cwlJ</i> spores using Method A
 +
|-
 +
| '''[https://2009.igem.org/Team:Newcastle/Labwork/19_August_2009#Sporulation_Tuning.2F_Chassis_Team 19/08/09]'''
 +
| Successful results for the treatment of the double-knockout mutants <i>sleB</i> and <i>cwlJ</i> spores using Method A
 +
|-
 +
| '''[https://2009.igem.org/Team:Newcastle/Labwork/25_August_2009#Chassis.2FSporulation_Team 25/08/09]'''
 +
| Freezing down of the treated non-germinating spores, <i>cwlD</i>, and <i>sleB</i> and <i>cwlJ</i>.
 +
|-
 +
| '''[https://2009.igem.org/Team:Newcastle/Labwork/2_September_2009#Chassis_team 02/09/09]'''
 +
| PCR-ing of gene <i>sleB</i> and <i>cwlJ</i> using primers previously designed and ordered.
 +
|-
 +
| '''[https://2009.igem.org/Team:Newcastle/Labwork/3_September_2009#Formal_Lab_Session_-_3rd_September_2009 03/09/09]
 +
| Attempt to PCR-ing of gene <i>sleB</i> and <i>cwlJ</i> using primers previously designed and ordered again.
 +
|-
 +
| '''[https://2009.igem.org/Team:Newcastle/Labwork/4_September_2009#Chassis_team 04/09/09]
 +
| Redesign PCR primers
 +
|-
 +
| '''[https://2009.igem.org/Team:Newcastle/Labwork/8_September_2009 08/09/09]
 +
| Cloning of sleB
 +
|}
==BioBrick constructs==
==BioBrick constructs==
 +
 +
'''BBa_K174012'''
 +
 +
''sleB'', ''Bacillus subtilis'' germination gene with RBS
 +
 +
Length: 932bp
 +
 +
[[Image:TeamNewcastleBBSleBandRBS.jpg|center|500px]]
 +
 +
 +
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
 +
 +
[[Image:TeamNewcastleBBCwlJandRBS.jpg|center|500px]]
 +
 +
 +
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
 +
 +
[[Image:TeamNewcastleBBSleBandcwlJ.jpg|center|100px]]
 +
 +
 +
Click [http://partsregistry.org/wiki/index.php?title=Part:BBa_K174014 ''here''] for more information on this part.
==Lab Work Strategies==
==Lab Work Strategies==
-
==Other Presentations and Diagrams==
+
[[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>==
 +
 
 +
[1] Popham, D., Helin, J., Costello, C & Setlow, P. (1996). Muramic lactam in peptidoglycan of <i>Bacillus subtilis</i> spores is required for spore outgrowth but not for spore rehydration or heat resistance. <i>Proc. Natl. Acad. Sci.</i> 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 <i>Bacillus subtilis</i> endospore germination. <i>Microbiology</i> 148; 2383-2392
 +
 
{{:Team:Newcastle/Footer}}
{{:Team:Newcastle/Footer}}
{{:Team:Newcastle/Right}}
{{:Team:Newcastle/Right}}

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.

... 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

Wet Lab

Click on the dates to go the the particular lab session.

Summary of Lab Sessions for Chassis
Date
Description
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

TeamNewcastleBBSleBandRBS.jpg


Click here for more information on this part.


BBa_K174013

cwlJ, Bacillus subtilis germination gene with RBS

Length: 441bp

TeamNewcastleBBCwlJandRBS.jpg


Click here for more information on this part.


BBa_K184014

cwlJ and sleB, Bacillus subtilis germination genes

TeamNewcastleBBSleBandcwlJ.jpg


Click here for more information on this part.

Lab Work Strategies

TeamNewcastleChassisFigure1.jpg

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




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