Team:Newcastle/Metals

=Metal Sequester= In our system, the team have used the metallothionein SmtA (which usually mops up zinc ions) and have linked it to the CotC spore coat protein - this means that, should the stochastic switch decide, any cadmium mopped up could be sequestered into spores thereby making it unavailable. Please click on the following links to learn more:
 * Introduction
 * Novelty in this sub-project
 * BioBrick constructs
 * Lab Work Strategies
 * Lab Work Done
 * References

Introduction
In order to take up and keep the cadmium from the soil efficiently, we needed to think about a way to cross-link the metal ions to intra-cellular proteins which would end up for the most of it in the spore. If the heavy metal ions were not cross linked to a "sponge protein", it could be lethal to the cell at lower concentration and the cell could end up sporulating too early, or even bursting in the soil, releasing all the heavy metal it has taken up. From our meeting with Prof. Nigel Robinson on the 18th of March 2009, it was evident that the best way of getting cadmium into the spore is to express a metallothionein, which would 'soak up' the cadmium, which in turn would be trapped within the protein in the spore.

Novelty in this sub-project
To increase the efficiency of our system in sequestering the heavy metal cadmium from the soil, we divised a plan to make sure that most of the metal ions that have been taken in our B.subtilis cell is then rendered bio-unavailable by incorporating it into smtA metallothionein. SmtA metallothionein protein from E. coli can bind to heavy metals [1,2,3]. They have a tendency to bind to cationic metal ions such as cadmium, copper, arsenic, mercury, silver. It has been shown that in B. subtilis, in order to express a specific protein in the spore coat, it is possible to make a fusion protein with a spore coat protein called CotC, and a group have successfully expressed antigen proteins which are about the same size as our smtA metallothionein in order to make a vaccine[4]. By fusing CotC spore coat protein from Bacillus subtilis, our smtA metallothionein can be localized to the spore coat, hence we hoped to successfully trap most of the metals ions into the bacterial spores. CotC was also fused with GFP reporter gene in order to detect the expression of the fusion protein into the spores using a fluorescence microscopy. Because we want most of the metals to go into the spore rather than the vegetative cell, we designed our fusion protein so that it is controlled by the native cotC promoter sigK which is activated in sporulation conditions. Therefore, our smtA metal sequester would only be expressed once the cell have made the decision to become a metal container and sporulate, and only be expressed in the spore. Finally, our construct integrated the B. subtilis genome through homologous recombination at the native CotC gene. However, as we will be using pMutin4 integration vector, there will be two different copies of the cotC gene in our engineered B.subtilis genome: a native copy of cotC and our engineered CotC-GFP-smtA fusion copy. We chose not to knock-out the native CotC gene because the lack of native CotC might lead to defects in spore coat formation.

BioBrick constructs


In the parts registry, BBa_K174008:



a) Construction
Synthesised by GeneArt, fragment: 1385 bp DNA

Clone Manager construct and cut map:

Sequencher construct:



b) Cloning and Integration
GeneArt cloned the vector into a standard biobrick vector. We sent them Plasmid pSB1AT3 with part BBa_J04450 (mCherry).

BBa_J04450 in pSB1AT3 Clone Manager plamid map:



Once we received this fragment cloned in pSBAT3 we amplified the part by PCR. Primer 1 will incorporate a HinDIII site and primer 2 will incorporate a BamHI. Checked that these enzymes don’t cut the fragment:



We cut the PCR fragment and clone into pMutin4 cut with the same enzymes (diagram below is pMutin2 but this is essentially the same)



We then integrate into the 168 chromosome using homology between our cotC fusion and the native copy of cotC.

c) Testing and Characterisation
Once transformed the spores of the mutant will need to be tested for fluorescence. Two obvious methods:

1)	Fluorescence microscopy: grow the cells in sporulation medium and look under the microscope for fluorescent spores. 2)	Purify spores and measure their fluorescence in a fluorescence plate reader. 3) We would also test whether the strain is able to absorb cadmium in the spores when compared to the wild-type.