Cadmium contamination can be a serious problem in countries where polluting industries are located close to agricultural sites. Our team developed a design to address this problem using the resiliant spore-forming bacterium Bacillus subtilis. We engineered B. subtilis to sense and sequester cadmium from the environment into metallothionein containing spores, rendering it bio-unavailable. Germination of the spores can be disabled, making retrieval of the cadmium unnecessary since they can persist intact for thousands of years. We computationally simulated the life cycles of individual cells and entire cell populations, to estimate the parameter values necessary to maintain sustainable populations of sporulating, germinating and vegetative cells. Our design required us to engineer stochastic differentiation processes at a single cell level. A sporulation rate tuner was developed and we also engineered a tuneable stochastic invertase switch to stochastically control cell differention and fate.

The following diagram gives an overview of what our design hopes to achieve...

Heavy Metal Sensing

Our design is for the bacteria to intake cadmium through the manganese channel MntH, as it has been found that due to the similar size and charge of the ions cadmium leaks through these channels [1].Once inside the cell the cadmium must be sensed in order for the cell to regulate some of the decisions involved in becoming a metal container. The metal sensing proteins we intend to use are yozA (czrA) and arsR which both bind cadmium as well as zinc cobalt and nickel, and arsenic, silver and copper respectively [2].These proteins are repressor proteins which also bind DNA preventing transcription of downstream CDS. The repressor proteins however release the DNA to preferentially bind cadmium [2] allowing transcription to occur. These proteins therefore allow selective sensing of cadmium in the form of an AND gate, a model for which can be seen in our modelling section.

Population Dynamics

As well as sensing cadmium we will attempt to engineer our bacteria’s normal population dynamics, by nudging the natural stochastic sporulation decision in favour of greater sporulation, to account for the spores that will be lost as metal containers. We will also design our own stochastic switch, which will be based on the decision of being a metal container or not. This will be regulated by an invertible segment of DNA using the hin/hix system [3]. In this way our artificial stochastic switch will be a ‘biased heads or tails’ which we can control. This stochastic switch will control expression of our ‘metal sponge’.

Metal Containers

To make our spores metal containers we will be using the metallothionein protein smtA which is a relatively small cysteine rich protein known to ‘soak up’ metals such as cadmium [4]. We will guide this protein to the spore whilst sporulating by creating a fusion with the spore coat protein cotC, which will coat the spore in cadmium bound metallothionein [5]. Finally, to ensure that metal containing spores will not germinate again, releasing their bound cadmium, we will ‘knock-out’ important germination genes using our invertible sequence system.

Novelty