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.
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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.
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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 <i>cwlD</i>, and <i>sleB</i> and <i>cwlJ</i>.
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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.
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Therefore, a mechanism is needed to allow us to choose to turn on germination, when the cell is not a "metal container".
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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-germination spores from Prof. Anne Moir, we performed lab experiments for the two non-germination spores, and concluded that the double-knockout mutant, <i>sleB</i> and <i>cwlJ</i> 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, <i>cwlD</i>.
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We propose that we could use IPTG as a switch for germination.
We propose that we could use IPTG as a switch for germination.
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===<i>Bacillus subtilis cwlD</i> mutant===
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:''[https://2009.igem.org/Team:Newcastle/Chassis/Introduction#Bacillus_subtilis_cwlD_mutant ... Click to read more ...]
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Together with the non-germination spores and [https://2009.igem.org/Team:Newcastle/Project/Labwork/MoreProtocols#Recovery_of_cwlD_spores protocol for recovery] which Prof. Anne Moir sent to us, information on the <i>cwlD</i> mutant was given as well, quoted in the following paragraph.
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==Novelty in this sub-project==
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"The <i>cwlD</i> (Cm<sup>r</sup>) mutation was transformed in our lab strain of <i>Bacillus subtilis</i> (1620) to create a mutant lacking muramic lactam in the cortex peptidoglycan. As this is a target for the activated cortex lytic enzymes, there is no cortex breakdown, limited rehydration, no gross protein changes in the spore coat, and hence the strain is germination defective."
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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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From the reference recommended by Prof. Anne Moir, we found that, the mutation in the <i>cwlD</i> gene of <i>Bacillus subtilis</i> is predicted to encode a muramoyl L-alanine amidase, which results in the production of spores containing no muramic lactam. These spores have normally dehydrated protoplasts but are unable to complete the germination/outgrowth process to produce viable cells.<sup>[1]</sup>
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Germination in the presence of lysozyme allows the <i>cwlD</i> spores to produce viable cells with normal heat resistance properties.<sup>[1]</sup>
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===<i>Bacillus subtilis sleB</i> and <i>cwlJ</i> double-knockout mutant===
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The <i>sleB</i> and <i>cwlJ</i> proteins are cortex-lytic enzymes, partially redundant in function, and are required together for effective cortex hydrolysis during <i>Bacillus subtilis</i> spore germination. Enzymic hydrolysis of spore-cortex peptidoglycan is essential for spores to complete rehydration during germination and to commence outgrowth,<sup>[2]</sup> thus the sleB and cwlJ proteins are important enzymes in normal spore germination of <i>Bacillus subtilis</i>.
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==Novelty in this sub-project==
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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>
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{| border="1"
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{| class=wikitable border="1"
|-
|-
! 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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==Lab Work Strategies==
==Lab Work Strategies==
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1. PCR up <i>sleB</i> and RBS using EcoRI and XbaI (Primer JJ1 – 5’) and SpeI (Primer JJ2 – 3’) as illustrated in Figure 1.
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[[Image:TeamNewcastleChassisFigure1.jpg|center|450px]]
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[[Image:TeamNewcastleChassisFigure1.jpg|center|350px|thumb|<center>Figure 1: RBS + ''sleB''</center>]]
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<u>Labwork:</u>
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1.1 Perform PCR with Primer JJ1 and Primer JJ2 on wild type Bacillus subtilis, where the <i>sleB</i> region will be amplified.
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1.2 Carry out DNA gel electrophoresis after the amplification of the DNA in Step 1.1, and we should see a fragment of approximately 918bp.
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2. Cut pSB1AT3 or pSB1A2 (BioBrick compatible vector, already have it in stock) with EcoRI and SpeI, then purify backbone fragment using kit to get rid of mCherry.
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[[Image:TeamNewcastleChassisFigure2.jpg|center|350px|thumb|<center>Figure 2: Cut mCherry out of the plasmid backbone (pSB1AT3 or pSB1A2)</center>]]
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[[Image:TeamNewcastleChassisFigure3.jpg|center|350px|thumb|<center>Figure 3: Remaining backbone fragment after cutting out mCherry</center>]]
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<u>Labwork:</u>
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2.1 Carry out restriction digest using the restriction enzymes EcoRI and SpeI, where the DNA segment mCherry is cut.
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2.2 The DNA segment is then analysed via gel electrophoresis where the shorter fragment would be mCherry, and the longer fragment, the backbone fragment.
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2.3 Use kit to process the backbone DNA fragment.
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[[Image:TeamNewcastleChassisFigure4.jpg|center|300px|thumb|<center>Figure 4: Plasmid part BBa_J04450 (pSB1AT3)</center>]]
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3. Ligate backbone fragment from Step 2, with PCR-ed sleB and RBS from Step 1, then cut with EcoRI and SpeI resulting in pJJ1.
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[[Image:TeamNewcastleChassisFigure5.jpg|center|350px|thumb|<center>Figure 5: Ligating backbone fragment and PCR-ed RBS and ''sleB''</center>]]
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[[Image:TeamNewcastleChassisFigure6.jpg|center|350px|thumb|<center>Figure 6: pJJ1 - Ligated backbone fragment and PCR-ed RBS and ''sleB''</center>]]
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4. PCR up <i>cwlJ</i> and RBS using EcoRI and XbaI as (Primer JJ3) and SpeI (Primer JJ4).
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[[Image:TeamNewcastleChassisFigure7.jpg|center|350px|thumb|<center>Figure 7: RBS and ''cwlJ''</center>]]
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<u>Labwork:</u>
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4.1 Perform PCR with Primer JJ3 and Primer JJ4 on wild type Bacillus subtilis, where the <i>cwlJ</i> region will be amplified.
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4.2 Carry out DNA gel electrophoresis after the amplification of the DNA in Step 4.1, and we should see a fragment of approximately 426bp.
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5. Purify and perform a midi prep for pJJ1 and cut with EcoRI and XbaI (restriction digest) to produce the fragment as seen in Figure 9.
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[[Image:TeamNewcastleChassisFigure8.jpg|center|350px|thumb|<center>Figure 8: Cutting pJJ1 with restriction enzymes ECoRI and XbaI</center>]]
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[[Image:TeamNewcastleChassisFigure9.jpg|center|350px|thumb|<center>Figure 9: pJJ1 fragment awaiting for the RBS and ''cwlJ'' fragment</center>]]
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<u>Labwork:</u>
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5.1 Transform E.coli with pJJ1.
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5.2 Conduct a mini prep.
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5.3 Carry out gel electrophoresis.
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5.4 Analyse results obtained from the gel electrophoresis.
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5.5 If result is correct, carry out a midi prep to obtain lots of DNA.
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5.6 Cut pJJ1 with restriction enzymes EcoRI and XbaI.
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6. Ligate the product from Step 5 with PCR-ed <i>cwlJ</i> and RBS which were cut with EcoRI and SpeI, resulting in pJJ2 as seen in Figure 11.
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[[Image:TeamNewcastleChassisFigure10.jpg|center|350px|thumb|<center>Figure 10: Ligating PCR-ed RBS and ''cwlJ'' with remaining pJJ1 fragment</center>]]
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[[Image:TeamNewcastleChassisFigure11.jpg|center|350px|thumb|<center>Figure 11: pJJ2 – Ligated PCR-ed RBS and ''cwlJ'' with pJJ1 fragment</center>]]
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7. Transform pJJ2, pick the correct colony and perform a mini prep to check
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<u>Labwork:</u>
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7.1 Transform E.coli with pJJ2.
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7.2 Conduct a mini prep.
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7.3 Carry out gel electrophoresis.
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7.4 Analyse results obtained from the gel electrophoresis.
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7.5 If result is correct, carry out a midi prep to obtain lots of DNA.
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8. PCR the joined up <i>sleB</i> and <i>cwlJ</i> from pJJ2 using HindIII (Primer JJ5) and (Primer JJ6) BamHI primers.
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[[Image:TeamNewcastleChassisFigure12.jpg|center|350px|thumb|<center>Figure 12: RBS and ''cwlJ'', and RBS and ''sleB'' fragment</center>]]
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;Labwork:
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8.1 Perform PCR with Primer JJ5 and Primer JJ6 on pJJ2, where the RBS + <i>cwlJ</i> and RBS +  <i>sleB</i> region will be amplified.
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8.2 Carry out DNA gel electrophoresis after the amplification of the DNA in Step 8.1, and we should see a fragment of approximately ___bp.
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;Result:
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[[Image:TeamNewcastleChassisFigure13.jpg|center|500px|thumb|<center>Figure 13: RBS and ''sleB'' sequence</center>]]
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[[Image:TeamNewcastleChassisFigure14.jpg|center|500px|thumb|<center>Figure 14: ''sleB'' cut sites</center>]]
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[[Image:TeamNewcastleChassisFigure15.jpg|center|500px|thumb|<center>Figure 15: RBS and ''cwlJ'' sequence</center>]]
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[[Image:TeamNewcastleChassisFigure16.jpg|center|500px|thumb|<center>Figure 16: ''cwlJ'' cut sites</center>]]
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;Cloning:
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9. Clone the joined up <i>sleB</i> and <i>cwlJ</i> from Step 8 into pMutin4 with HindIII and BamHI primers.
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[[Image:TeamNewcastleChassisFigure17.jpg|center|500px|thumb|<center>Figure 17: Ligating the RBS and ''cwlJ'', and RBS and ''sleB'' segment into pMutin4</center>]]
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[[Image:TeamNewcastleChassisFigure18.jpg|center|300px|thumb|<center>Figure 18: pMutin4</center>]]
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10. PCR pSpac:<i>cwlJ</i>:<i>sleB</i> from pMutin 4 with suitable primers for insertion into pGFP-rrnB. Suitable primers being EcoRI and XbaI (Primer PJJ7) and SpeI and PstI (Primer PJJ8).
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[[Image:TeamNewcastleChassisFigure19.jpg|center|350px|thumb|<center>Figure 19: pSpac:cwlJ:sleB fragment</center>]]
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[[Image:TeamNewcastleChassisFigure20.jpg|center|300px|thumb|<center>Figure 20: ''pGFP-rrnB''</center>]]
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11. Integrate
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;Testing and Characterisation:
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We intend to use IPTG at difference concentrations to induce the promoter pSpac.
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==Other Presentations and Diagrams==
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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.

... 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 [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

TeamNewcastleBBCwlJandRBS.jpg


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

TeamNewcastleBBSleBandcwlJ.jpg


Click [http://partsregistry.org/wiki/index.php?title=Part:BBa_K174014 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




News

Events

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]
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