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Our Project

The aim of our project is to genetically engineer Bacillus subtilis to be able to detect and sense cadmium which has been taken up from the soil environment and bound to metallothionein. This metallothionein will then become incorporated into a Bacillus spore, the resilience of which means that the cadmium ions can become isolated from the environment (and made bio-unavailable) for many years.

This project involves a number of steps, each of which can be considered as sub projects:

1. Cadmium Intake
2. Cadmium Sensing
3. Cadmium sequestration by metallothionein
4. Sporulation Tuning
5. Chassis
6. Population simulation
7. Stochastic Switch
8. Synthesizing a Promoter Library for Bacillus subtilis

Cadmium Intake

For this project we want to be able to process cadmium and cadmium only. 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 (from the 168 strain) takes up cadmium naturally through the manganese transport system (Laddaga. R.A., Bessen. R., Silver. S.; 1985). Additionally it has been demonstrated that mutations to the manganese transport system can affect its ability to uptake cadmium without affecting its ability to transport manganese (Zeigler. D.R., et al; 1987).

The manganese ion channel that we intend to either upregulate or control is the mntH ion channel (also goes under the name ydaR). mntH is part of the Nramp family of proton-coupled metal ion transporters (Que. Q., Helmann. J.D.; 2000). It is also classified as a secondary transporter (Membrane Transport website).

It is also known that mntH is regulated negatively by increasing manganese ion concentrations. This is allowed to happen through the promoter called mntR (Que. Q., Helmann. J.D.; 2000). This means that B.subtilis has the ability to limit the manganese metal intake system when the intracellular concentrations of Mn2+ starts to approach cytotoxic levels.

Cadmium Sensing

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.

There are two metal sensing repressors, which are known to respond to cadmium: ArsR and CzrA (also known as YozA).

i) ArsR

ArsR (also known as YqcJ) is a protein which is part of the ArsR-SmtB family of transcriptional regulators. It is a regulating protein for the arsenic resistance operon in Bacillus subtilis (NCBI website – arsR family transcription regulator profile) (Moore. C.M and Helmann. J.D; 2005).

The ArsR protein acts as a repressor until it is conformationally changed by the presence of arsenic ions (Harvie. D.R, et al; 2006). However, arsenic is not the only metal which can cause this action to happen; it has been noted that silver (Ag(I)), cadmium (Cd) and copper (Cu) (Moore. C.M and Helmann. J.D; 2005) can cause this action to happen.

Metal Sensor	Metals Sensed
ArsR	As(III)	Ag(I)	Cu	Cd

ii) CzrA (YozA)

CzrA (also known as YozA) is a member of the ArsR-SmtB family of transcriptional regulators (Moore. C.M and Helmann. J.D; 2005)(Harvie. D.R, et al; 2006). Like ArsR, it is a regulator protein which can be relieved from binding to the DNA by being bound to by metal ions – these include zinc (Zn), cobalt (Co), nickel (Ni) and cadmium (Cd) (Moore. C.M and Helmann. J.D; 2005). This can be summarised in the table below:

Metal Sensor	Metals Sensed
czrA	Zn	Co	Ni	Cd

AND Gating

If the two ion selectivity tables are put together, it can be seen that the metal common to both sensors is cadmium:

Metal Sensor	Metals Sensed
ArsR	As(III)	Ag(I)	Cu	Cd
CzrA	Zn	Co	Ni	Cd

This means that if the two repressors can act on a single promoter or binding site by AND gating, cadmium can be detected and a biological response can be triggered.

The presence of cadmium ions releases both ArsR and CzrA binding proteins from the DNA allowing transcription to occur.

Cadmium sequestration by metallothionein

So the cadmium has made its way into the cell; and the cadmium has been detected by the metal sensors arsR and czrA. The question is: what happens to the cadmium now?

In our project, we hope to soak up the intracellular cadmium with a metallothionein known as smtA. Metallothioneins are proteins which have great tendencies to bind to cationic metal ions; examples of which include copper (Cu), zinc (Zn), lead (Pb) and cadmium (Cd) (Creti. P., et al; 2009). This property is due to the richness of cysteine residues in its structure (Creti. P., et al; 2009).

In the cell, metallothioneins generally have two important roles: to remove non-essential metals via sequestration and to control levels of essential metals (Creti. P., et al; 2009). It is the first role with which we are concentrating on.

smtA encodes a metallothionein of the same name – this protein, which is described as a class II metallothionein seems to be synthesized in response to metals such as zinc and cadmium (Morby. A.P., et al; 1993). This suggests it has a role in cadmium sequestration.

Our Bacillus spores are coated in the metallothionein by making a fusion protein with cotC a major spore coat protein, our metal sponge should locate to the spore making the cadmium bio-unavailable.

Stochastic switch

We developed a tuneable, heritable stochastic switch to stochastically control cell differention and fate. This switch encodes a heads or tails device that can be biased using two inducible promoters and by controlling the rate of degradation of the protein responsible for the switching, Hin recombinase.

In our project we used the switch to make a decision for cells to be a metal container and sequester cadmium, or continue to normal vegetative life. This cell fate decision is given based on a number of stochastic parameters. Hin Recombinase is a protein from Salmonella that is able to invert a segment of DNA between two inverted repeats of DNA (Hix sites). The Hin recombinase CDS is also included in this flipping region. This promoter's activity and direction determines the cells' fate. We carried out stochastic modelling of the system in order to fine tune it to produce a 'biased heads or tails' switch.

The unique part about our stochastic device is that it is controlled in three ways; 2 variable strength promoters (which we tested using inducible promoters Pspac and XylR), and a degradation controller sspB, and so tuning of Hin expression (and thus flipping rate) can be tightly controlled. We have modelled this degradation control, which we believe is novel within the iGEM competition.

The following diagram shows the device either BEFORE flipping has taken place, or after the stochastic decision has determined it to be in this orientation...

Therefore the germination genes are complemented and we get bacillus cells germinating and carrying out a vegetative life cycle, whilst extra cadmium efflux channels are expressed to ensure cell survival.

The following diagram shows the device afer the stochastic decision has taken place, orienting the flipping region to the right...

Therefore extra KinA is expressed which phosphorylates the master regulator protein Spo0A, activating it and increasing sporulation rate. As well as this, the CotC-Metallothionein (smtA) fusion is expressed, which 'soaks up' cadmium and locates it to the developing spore.

We developed the following bricks associated with the stochastic switch and entered them onto the parts registry:

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K174003 BBa_K174003] Heritable, Tunable, Stochastic Switch

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K174002 BBa_K174002] Arabinose controlled protein degradation device

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K174000 BBa_K174000] SspB proteolysis chaperone

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K174001 BBa_K174001] Arabinose inducible system

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K174006 BBa_K174006] Sac single crossover site for Bacillus subtilis

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K174007 BBa_K174007] Degradation controller with integration site

Sporulation Tuning

The bacteria, Bacillus subtilis, used in our project is a gram-positive soil bacterium that, under certain conditions, would commit itself to a developmental pathway leading to the production of spores.(Predich, M., et al; 1992) Therefore, in this section of our project, we hope to control sporulation in our bacterial population, such that we can decide how much of the population becomes spores, and how much continue as vegetative cells. Should the cell sporulate, it would become a ‘metal container’, trapping the sequestered cadmium in its spore.

After the cell sequesters cadmium into its spore, it should not germinate or the sequestered cadmium will be released back into the environment as a result. Therefore, the role of chassis comes into play, where the sleB and cwlJ germination-defective mutants are put into use.

In order to control sporulation, our team is proposing the idea of inducing the synthesis of KinA, with IPTG as a sporulation initiation signal.

Chassis

Since the main aim of our project is to sequester cadmium in the environment into the spores of our engineered B. subtilis, but what happens after the cadmium has been sequestered?

Do we attempt to retrieve the sequestered cadmium? Or, do we simply leave the sequestered cadmium in the spores of our engineered B. subtilis?

For our project, we have chosen the latter. We will not be attempting to retrieve the sequestered cadmium. However, then comes the question of, would there not be chances of the cadmium entering the environment again?

Our solution to this question would be to disable germination of the spores, thus retrieval of the sequestered cadmium becomes unnecessary, as the spores can persist intact for thousands of years.

While we would like to disable germination for the spores that contain sequestered cadmium, not all the cells would have sequestered cadmium, and it is also essential that we still have some cells germinating, so that our population of bacteria can continue to live and grow, reaching a balance, and not simply deplete totally.

Therefore, a mechanism is needed to allow us to choose to turn on germination, when the cell is not a "metal container". In order to do so, our team intends to use IPTG as a switch for germination, and the non-germination spores used in our project is the double-knock out mutant, sleB and cwlJ.

Population simulation

Due to our project being based around taking a section of our bacterial population, and making them sporulate, but not germinate again, we need to make sure that we do not kill off our entire population. Thus we need to be able to tune our system, so that we can have a large enough percentage of metal sequestering spores to make a positive environmental impact, but also a small enough percentage, so that the population will continue to live and grow.

The novel part of this sub-project is to model the dynamics of a bacterial population, on the cellular level, as well as integrating this agent based model with biochemical models. Furthermore our simulation is able to run on distributed systems, making use of a large number of computers at once.

This section of the project is a model simulation, which describes a high level working of our complete system. It also includes detail from other models, as it uses these in its decision making processes. The model has been developed in the Java programming language, but also uses other technologies including running CellML models.

Promoter Library

Some of the promoters we intend to use for our BioBricks will require different strengths. Part of our project will involve making a library of Sigma A promoters for Bacillus subtilis and measuring their varying strengths; with this data we will use a sigma A promoter variant for a BioBrick according to the desired strength. As stated in the instructors meeting on the 22/04/09, we could measure the expression of fluorescent protein.

We designed variances of sigA consensus sequence by degenerating it between and around -35 region and -10 region.

References

Membrane Transport website (http://www.membranetransport.org/all_type_btab.php?oOID=bsub1 )

Charles, M. M., G. Ahmed, et al. (2005). "Genetic and physiological responses of Bacillus subtilis to metal ion stress." Molecular Microbiology 57(1): 27-40.

Cretì, P., F. Trinchella, et al. "Heavy metal bioaccumulation and metallothionein content in tissues of the sea bream Sparus aurata from three different fish farming systems." Environmental Monitoring and Assessment.

Duncan, R. H., A. Claudia, et al. (2006). "Predicting metals sensed by ArsR-SmtB repressors: allosteric interference by a non-effector metal." Molecular Microbiology 59(4): 1341-1356.

Laddaga, R. A., R. Bessen, et al. (1985). "Cadmium-resistant mutant of Bacillus subtilis 168 with reduced cadmium transport." J. Bacteriol. 162(3): 1106-1110.

Moore, C. M. and J. D. Helmann (2005). "Metal ion homeostasis in Bacillus subtilis." Current Opinion in Microbiology 8(2): 188-195.

Morby, A. P., J. S. Turner, et al. (1993). SmtB is a metal-dependent repressor of the cyanobacterial metallothionein gene smtA: identification of a Zn inhibited DNA-protein complex. 21: 921-925.

Qiang, Q. and D. H. John (2000). "Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins." Molecular Microbiology 35(6): 1454-1468.

Zeigler, D. R., B. E. Burke, et al. (1987). "Genetic mapping of cadmium resistance mutations inBacillus subtilis." Current Microbiology 16(3): 163-165.

Predich, M., Nair, G., Smith, I. (1992) Bacillus subtilis Early Sporulation Genes kinA, spo0F, and spo0A Are Transcribed by the RNA Polymerase Containing σH. Journal of Bacteriology. Pp 2771-2778