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Metal Sequester

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 can successfully trap most of the metals ions into the bacterial spores.

It is 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 will only be expressed once the cell have made the decision to become a metal container and sporulate, and it will only be expressed in the spore.

BioBrick constructs

Lab Work Strategies

Other Presentations and Diagrams

References

1- Cretì, P., F. Trinchella, et al. "Heavy metal bioaccumulation and metallothionein content in tissues of the sea bream Sparus aurata from three different fish farming systems." Environmental Monitoring and Assessment.

2- Morby, A. P., J. S. Turner, et al. (1993). SmtB is a metal-dependent repressor of the cyanobacterial metallothionein gene smtA: identification of a Zn inhibited DNA-protein complex. 21: 921-925.

3- Waldron, K. J. and N. J. Robinson (2009). "How do bacterial cells ensure that metalloproteins get the correct metal?" Nat Rev Micro 7(1): 25-35.

4- Mauriello, E. M. F., L. H. Duc, et al. (2004). "Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner." Vaccine 22(9-10): 1177-1187.