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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.

Project Description

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: <img src="" width="590" height="442" border="0" usemap="#Map" /> <map name="Map" id="Map">

 <area shape="rect" coords="284,101,441,170" href="HIN" /><area shape="rect" coords="351,180,559,259" href="KINA" />
 <area shape="rect" coords="321,265,492,346" href="COTC" /><area shape="rect" coords="206,197,307,315" href="SS" /><area shape="rect" coords="104,261,188,342" href="GERM" />
 <area shape="rect" coords="5,230,93,359" href="CADA2" /><area shape="rect" coords="24,114,79,172" href="MNTH" />

<area shape="rect" coords="96,82,139,143" href="CADA" /> <area shape="rect" coords="146,117,238,186" href="ARSR" /><area shape="rect" coords="81,153,145,212" href="CZRA" /> <area shape="rect" coords="157,9,295,87" href="CADMIUM" /> <area shape="rect" coords="374,28,580,91" href="SUBTILIS" /> </map> <img src=" width="550" height="332" border="0" usemap="#Map" /> <map name="Map" id="Map"><area shape="rect" coords="7,25,246,195" href="VEG" /> <area shape="rect" coords="293,17,540,180" href="FORE" /> <area shape="rect" coords="336,183,526,328" href="SPORE" />

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.

References

1. Que, Q. and J.D. Helmann, Manganese homestasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Molecular Microbiology, 2000. 35(6): p. 1454-1468.
2. Harvie, D.R., et al., Predicting metals sensed by ArsR-SmtB repressors: Allosteric interference by a non-effector metal. Molecular Microbiology, 2006. 59(4): p. 1341-1356.
3. Haynes, K.A., et al., Engineering bacteria to solve the Burnt Pancake Problem. Journal of Biological Engineering, 2008. 2.
4. Blindauer, C.A., et al., Multiple bacteria encode metallothioneins and SmtA-like zinc fingers. Molecular Microbiology, 2002. 45(5): p. 1421-1432.
5. Mauriello, E.M.F., et al., Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner. Vaccine, 2004. 22(9-10): p. 1177-1187.

External links

• [http://www.ncl.ac.uk Newcastle University]