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. Using the smtA metallothionein from E.coli and the CotC coat protein as a fusion partner, the metallothionein could in theory be localised to the spore coat as it forms (under control of mother cell specific SigK).

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. 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 bacterial spores. It is also fused with Gfp to see the spores using a microscope. The fusion protein expression will be controlled by sigK promoter which is activated by sporulation conditions.

We engineered the smtA gene so that it is fused with the spore coat protein cotC and the reporter gene GFP. This fusion will ensure that most of the smtA expressed will be attached to the spore coat and therefore the heavy metal ions that are incorporated into the smtA protein will be localised into the spore.

Team:Newcastle/Metals

From 2009.igem.org

Metal Sequester

Introduction

Novelty in this sub-project

Modelling

BioBrick constructs

Lab Work Strategies

Other Presentations and Diagrams