Team:Newcastle/Metalsensing

=Metal Sensing= This sub-project focusses on getting the Bacillus subtilis to sense the cadmium and being able to respond to this detection. Please click on the links below for more information:
 * Introduction
 * Modelling
 * BioBrick Constructs
 * Lab Work Strategies
 * Other Presentations and Diagrams
 * Lab Work done

Introduction
If our project is to process cadmium and not other metals, we need to genetically engineer Bacillus subtilis to carry out a set of cellular processes based on the action of metal sensors. These metal sensors will detect cadmium through a system known as AND Gating. This system of combined metal sensors ensures the Bacillus subtilis cells have a tightly controlled cadmium sensor, as naturally occurring metal sensitive promoters can respond to OTHER metals too

There are two metal sensing repressors, which are known to respond to cadmium: ArsR and CzrA. However, their specificity for cadmium is not unique. ArsR also detects arsenic and CzrA also detects zinc and copper. The table below shows the metals which release both ArsR and CzrA from their DNA binding sites: By positioning the operator binding sites for these two metal sensing repressors next to each other in a promoter region, the gene regulated by that promoter will be transcribed only when a metal that binds to both sensors is present; in this case Cadmium.

This is a combinatorial approach for gene expression regulation. To read more information on the cadmium sensor, please visit the Cadmium Sensing section of the Project Overview.

To create this AND gate the team made use of the CadA promoter; this promoter, which usually regulates expression of the CadA cadmium efflux channels in the presence of cadmium ions, bears the CzrA binding site. Therefore if this promoter is coupled to the ArsR binding site, expression of a signal protein will only be made in the presence of cadmium.

Why don't we just use the CadA promoter then? The answer is this - cadA can respond to other metals too so a system totally reliant on the cadA promoter as a sensor would not be efficient in achieving the aims of this project. We need a tightly-controlled cadmium sensor

Modelling
To simulate the effects of cadmium intake and sensing, the Metal Sensing Team constructed models using CellML. Please click on the link here: Metal Intake/Efflux Model

BioBrick constructs
For our cadmium-sensing BioBrick design, we have ligated the ArsR binding site(BBa_K174016) next to the CadA promoter (BBa_K174017). The CadA promoter drives expression of the CadA efflux channel and this expression activity increase in the presence of cadmium. Yet more importantly the CadA promoter region contains the CzrA binding site the team needs for the cadmium-sensor AND gate.

By placing the ArsR binding site next to the CadA promoter, expression of a signal protein will only be achieved if there is cadmium in the cell.

Lab Work Strategies
In carrying out the lab work required for the construction of the cadmium-sensor, we adhered to the following lab strategy. Please click on the link here to review our strategy: Metal Sensor: czrA+arsR.

Other Presentations and Diagrams
The diagram below illustrates the way in which ArsR and CzrA respond to cadmium ions, which will make their way into the cell via the MntH (manganese transporter) channel: Please refer the key below to better understand this diagram! In short, cadmium ions make their way through the MntH channel and bind to the ArsR and CzrA repressor proteins. Their removal allows the DNA polymerase to make a signal protein which will feed into the stochastic switch.

Lab Work done
This is all of the lab work in which the Metal Sensing Team attempted to construct the cadmium sensor - click on the dates to read that day's lab entry: