Team:Newcastle

= GOLD Medal winners 2009 =


 * In 2009 the Newcastle team are tackling environmental issues using Bacillus subtilis. We are a team of eight with wide ranging backgrounds in the fields of Bioinformatics, Computing Science, Chemical Engineering, Genetics and Medical Sciences. This is Newcastle's second year in the iGEM competition; last year our team designed BugBuster, which achieved a Gold Medal.


 * In 2009 we matched our Gold medal from the previous year, and hope to be back in 2010 to participate in iGEM again.

= Project Description = 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 our design, click on different areas of the diagram to go to the appropriate section of our project:

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Cadmium Sensing
Our design allows bacteria to intake cadmium through the manganese channel MntH, as cadmium also leaks through these channels in addition to other metals [1]. The metal sensing proteins we intend to use are 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 allowing selective sensing of cadmium in the form of a logic AND gate.

Population Dynamics
As well as sensing cadmium we plan to engineer our bacteria’s normal population dynamics, by nudging the natural stochastic sporulation decision in favour of higher sporulation rates, to account for the spores that will be lost as metal containers. Our population model simulates whole cell populations using inputs from single cellular models and will be used to determine the required increase in sporulation rate.

Stochastic switch
Our artificial stochastic switch in a ‘biased heads or tails’ which we can tune. The stochastic switch determines the cell's decision to become a metal container or not. This switch will be regulated by an invertible segment of DNA using the hin recombinase/hix [3]. We favoured the use of an invertable DNA segment since the choice will be heritable and maintain its expression inside a spore. This stochastic switch will either induce the expression of the genes which switch on the ‘metal sponge’ phenotype or will direct the cell to a wild-type lifestyle.

Metal Containers
To make our spores into 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 by creating a fusion with the spore coat protein CotC, which will embed cadmium bound metallothionein throughout the spore [5]. Finally, to ensure that metal containing spores will not germinate again, releasing their bound cadmium, we will ‘knock-out’ essential germination genes sleB and cwlJ and selectively re-complement the resulting mutation.

Sporulation tuning
In order to tune sporulation, our team is proposing the idea of inducing the synthesis of KinA, with IPTG as a sporulation initiation signal. KinA is a major kinase which provides phosphate input to the phosphorelay, which in turn, activates the sporulation pathway upon starvation via the phosphorylated Spo0A transcription factor,[2] which governs entry into the sporulation pathways of the bacterium Bacillus subtilis.[3]

Novelty
Our design has many components which we believe are novel, such as our control over the sporulation cycle, and our synthetic stochastic switch, both systems we believe are reusable concepts within synthetic biology.