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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 of, 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.

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, 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.

Therefore, a mechanism is needed to allow us to choose to turn on germination, when the cell is not a "metal container".

Using the 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, 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.

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

Bacillus subtilis cwlD mutant

Together with the non-germination spores and protocol for recovery which Prof. Anne Moir sent to us, information on the cwlD mutant was given as well, quoted in the following paragraph.

"The cwlD (Cm^r) mutation was transformed in our lab strain of Bacillus subtilis (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."

From the reference recommended by Prof. Anne Moir, we found that, the mutation in the cwlD gene of Bacillus subtilis 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.^[1]

Germination in the presence of lysozyme allows the cwlD spores to produce viable cells with normal heat resistance properties.^[1]

Bacillus subtilis sleB and cwlJ double-knockout mutant

The sleB and cwlJ proteins are cortex-lytic enzymes, partially redundant in function, and are required together for effective cortex hydrolysis during Bacillus subtilis spore germination. Enzymic hydrolysis of spore-cortex peptidoglycan is essential for spores to complete rehydration during germination and to commence outgrowth,^[2] thus the sleB and cwlJ proteins are important enzymes in normal spore germination of Bacillus subtilis.

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.

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

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

1. PCR up sleB and RBS using EcoRI and XbaI (Primer JJ1 – 5’) and SpeI (Primer JJ2 – 3’) as illustrated in Figure 1.

Labwork:

1.1 Perform PCR with Primer JJ1 and Primer JJ2 on wild type Bacillus subtilis, where the sleB region will be amplified. 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.

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.

Labwork:

2.1 Carry out restriction digest using the restriction enzymes EcoRI and SpeI, where the DNA segment mCherry is cut. 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. 2.3 Use kit to process the backbone DNA fragment.

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.

4. PCR up cwlJ and RBS using EcoRI and XbaI as (Primer JJ3) and SpeI (Primer JJ4).

Labwork:

4.1 Perform PCR with Primer JJ3 and Primer JJ4 on wild type Bacillus subtilis, where the cwlJ region will be amplified. 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.

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.

Labwork:

5.1 Transform E.coli with pJJ1. 5.2 Conduct a mini prep. 5.3 Carry out gel electrophoresis. 5.4 Analyse results obtained from the gel electrophoresis. 5.5 If result is correct, carry out a midi prep to obtain lots of DNA. 5.6 Cut pJJ1 with restriction enzymes EcoRI and XbaI.

6. Ligate the product from Step 5 with PCR-ed cwlJ and RBS which were cut with EcoRI and SpeI, resulting in pJJ2 as seen in Figure 11.

7. Transform pJJ2, pick the correct colony and perform a mini prep to check

Labwork:

7.1 Transform E.coli with pJJ2. 7.2 Conduct a mini prep. 7.3 Carry out gel electrophoresis. 7.4 Analyse results obtained from the gel electrophoresis. 7.5 If result is correct, carry out a midi prep to obtain lots of DNA.

8. PCR the joined up sleB and cwlJ from pJJ2 using HindIII (Primer JJ5) and (Primer JJ6) BamHI primers.

Labwork

8.1 Perform PCR with Primer JJ5 and Primer JJ6 on pJJ2, where the RBS + cwlJ and RBS + sleB region will be amplified. 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.

Result

Cloning

9. Clone the joined up sleB and cwlJ from Step 8 into pMutin4 with HindIII and BamHI primers.

10. PCR pSpac:cwlJ:sleB 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).

11. Integrate

Testing and Characterisation

We intend to use IPTG at difference concentrations to induce the promoter pSpac.

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