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Metal Intake/Efflux

Introduction

The first step in our project is the uptake of the heavy metal cadmium (and cadmium only) into our B.subtilis cell. Therefore it would be logical to find a way in which we can increase the intake of cadmium without increasing the intake of other metals too.

It is known that a Bacillus subtilis cell takes up cadmium naturally through the manganese transport system. The manganese ion channel that we intend to either upregulate or control is the MntH ion channel. MntH is part of the Nramp family of proton-coupled metal ion transporters. MntH is regulated negatively by increasing manganese ion concentrations. This regulation ocurs via the promoter MntR. B.subtilis therefore has the ability to limit the manganese metal intake system when the intracellular concentration of Mn2+ starts to approach cytotoxic levels.

In B. Subtilis cation efflux is by an antiporter: the CadA efflux system. Cd, Co, Zn are pumped out by this ATPase dependant member of the CDF family.

We need to engineer our B.subtilis to be able to upregulate cadmium uptake and downregulate its efflux system in order to accumulate cadmium in the cell once the metal container decision has been made.

Modelling

• This is how the metal influx/efflux system works in isolation:
  

• When the stochastic switch is giving feedback from its metal container decision, this is how the system is engineered to work:

• Here are some graphs which the produced by our cellML model

BioBrick constructs