Newcastle iGEM Presentation 2009


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  • The stochastic switch sub project involves a flippable promoter region controlled by the Hin invertase protein. The region will undergo random flipping when the invertase is expressed. The invertase is under the control of 2 variable strength promoters as well as another protein ( sspb from E.coli ) which controls Hin’s degradation rate. This means that we have a 3 way control over the levels of the invertase and so over the flipping rate. Thus our switch is tuneable.
  • Here is an overview diagram of the entire device that we had synthesised. The diagram shows that for testing purposes we actually replaces the variable strength promoters with inducible promoters pSpac and xylA (induced by IPTG and Xylose respectively). Our degratation controller is induced by Arabinose using the Ara promoter region form the Bacillus ara operon. Overall this means that we are able to test this construct alone without the many other regulatory devices involved. And once characterised we can change the promoters due to the cut sited entered at either side of the inducible promoters.
  • We need to change these graphs to something that fits with what we have done.
  • Newcastle iGEM Presentation 2009

    1. 1. iGEM 2009 Team:Newcastle Bac-Man: The Quest to Sequester
    2. 3. We have a problem… <ul><li>… Cadmium </li></ul><ul><li>A white, silvery heavy metal </li></ul><ul><li>A group 12 transitional metal </li></ul><ul><li>Trace element </li></ul><ul><li>Danger to the environment! </li></ul><ul><li>Neurotoxic agent – Itai-Itai disease </li></ul><ul><li>Causes renal damage </li></ul><ul><li>Induces bone demineralisation </li></ul><ul><li>Cancer? </li></ul>Overview So what’s the problem?... … ACCUMULATION
    3. 4. What can our Project do about it? AIMS <ul><li>Detect cadmium in the environment and remove it from the environment rendering it bio-unavailable </li></ul><ul><li>Target cadmium at the source to avoid the damaging effects of accumulation. </li></ul><ul><li>Remodel the life cycle of Bacillus subtilis to accommodate it’s new role and to interact with the environment. </li></ul>
    4. 5. Our System <ul><li>Choice of organism = Bacillus subtilis : </li></ul><ul><li>Can produce resilient, long-lasting spores </li></ul><ul><li>Possibility of epi-chromosomal gene transfer </li></ul><ul><li>Naturally lives in soil </li></ul>Cadmium Intake Cadmium sequestering and sporulation Stochastic Switch Cadmium Sensing Cadmium efflux
    5. 6. <ul><li>SUB-PROJECTS INVOLVED IN OUR DESIGN : </li></ul><ul><li>Metal Infflux/Efflux </li></ul><ul><li>Metal Sensor </li></ul><ul><li>Stochastic Switch </li></ul><ul><li>Sporulation Tuner </li></ul><ul><li>Metal Sequester </li></ul><ul><li>Population Modelling </li></ul><ul><li>Chassis </li></ul>Sub-projects Overview
    6. 7. Cadmium Sensing
    7. 8. Cadmium Sensing: What is this sub-project about? <ul><li>AIM – we need to produce a tightly regulated cadmium sensor in our system which produces a signal in response </li></ul><ul><li>Need to design a promoter regulated by cadmium and responsive to the stochastic switch’s decision </li></ul><ul><li>Determines: </li></ul><ul><ul><li>The fate of a cell according to the environement </li></ul></ul><ul><ul><li>Feeds into the stochastic switch </li></ul></ul><ul><ul><li>Germination ability of spores </li></ul></ul><ul><li>How do we build our cadmium sensor BioBrick: </li></ul><ul><ul><li>Create an AND gate </li></ul></ul><ul><ul><li>Use metal sensors CzrA and ArsR </li></ul></ul>Metal Sensor
    8. 9. Cadmium Sensing: ArsR and CzrA <ul><li>Both are metal sensitive repressors: </li></ul><ul><ul><li>ArsR features in Arsenic Resistance operon </li></ul></ul><ul><ul><li>CzrA features in Cobalt zinc Resistance operon </li></ul></ul><ul><li>Why use these two metal sensors? </li></ul>For the same reason we are using the AND gate – metal sensitive promoters can sense MORE THAN ONE METAL! We know that the CadA promoter contains CzrA binding site Solution: engineer ArsR binding site next to CadA promoter to create cadmium sensing AND gate Metal Sensor Metals Sensed ArsR As(III) Ag(I) Cu Cd Metal Sensor Metals Sensed czrA Zn Co Ni Cd
    9. 10. Cadmium Sensing: BioBrick Construct AND gate BioBrick ArsR binding sites cadA promoter with CadA binding site RBS Metal Sensor
    10. 11. Cadmium Sensing: AND gate
    11. 12. Cadmium Sensing: Modelling Metal Sensor
    12. 13. Cadmium Sensing: Novelty and Importance <ul><li>Need tightly-controlled cadmium metal sensor – single metal sensors not enough. </li></ul><ul><li>Usual metal-sensitive promoters are sensed by a single repressor protein only </li></ul><ul><li>Our metal-sensitive promoter is regulated by 2 different repressor proteins: CzrA and ArsR </li></ul><ul><li>Can be used in front of any gene, not only in our project but in different settings. </li></ul><ul><li>The concept of using two metal sensors in this way can be adapted for sensing other metals too. </li></ul>Metal Sensor
    13. 14. Stochastic switch
    14. 15. Stochastic Switch: What is this sub-project? <ul><li>Tuneable invertible promoter region which </li></ul><ul><li>Determines: </li></ul><ul><ul><li>Rate of Bacillus sporulation </li></ul></ul><ul><ul><li>Metal sponge expression </li></ul></ul><ul><ul><li>Germination ability of spores. </li></ul></ul><ul><li>Degradation controller </li></ul><ul><li>This subproject allows us to control key aspects of the Bacillus life cycle to suit our projects needs all in a tuneable manner. </li></ul>Stochastic Switch
    15. 16. Stochastic Switch: Novelty in design <ul><li>The stochastic switch is the most novel part of our design </li></ul><ul><li>The switch regulates the decision to become a non-germinating metal container spore, or a spore that can go on to germinate as part of the normal life cycle </li></ul><ul><li>Hin invertase is used to switch on and off promoters. </li></ul><ul><li>We have added new bricks to our device to make the switch tuneable in three ways; 2 variable strength promoters and a degradation controller. </li></ul><ul><li>The switch controls 3 main aspects of the cell life cycle: </li></ul><ul><ul><li>Upregulating sporulation </li></ul></ul><ul><ul><li>Downregulating germination </li></ul></ul><ul><ul><li>Metallothionein-cotC fusion expression </li></ul></ul>Stochastic Switch
    16. 17. Stochastic Switch: BioBricks and Devices… EcoRI Xbal Biobrick Prefix Rfp AccIII BalI hixC hixC hin invertase sigA(pveg) pspac NaeI xlyA promoter NruI SpeI PstI Biobrick Suffix RBS + GFP Prefix (ATG) NheI Key Restriction site Invertase site Double terminator RBS ‘ Right facing’ promoter ‘ Left facing’ promoter <ul><li>The device we had synthesised was for testing purposes an so contained IPTG and Xylose inducible promoters </li></ul><ul><li>The hin invertase has a degradation tag recognised by degradation controller ssbp from E.coli. </li></ul><ul><li>Sspb is placed under the control of an arabinose inducible promoter. </li></ul>Stochastic Switch
    17. 18. Stochastic Switch: Modelling <ul><li>The device had to be modelled due to the many variables that contribute to the stochastic decision; these variables can be changed to give different pulse lengths of hin in order to give a net number of flips : </li></ul>Stochastic Switch
    18. 19. pSpac XylA Germination activator (FimE) Sporulation activator (CinR) pSpac XylA Sporulation activator (CinR) Germination activator (FimE) Hin <ul><ul><li>Generally: Pveg faces Left = germination </li></ul></ul><ul><li> Pveg faces Right = metal container decision. </li></ul>Stochastic Switch Stochastic Switch: Modelling hixC hixC hin Pveg hixC hixC hin Pveg
    19. 20. Cadmium Sequestration
    20. 21. <ul><li>AIM – to render cadmium bio-unavailable by mopping it up using a metallothionein and moving it into spores </li></ul><ul><li>By wrapping a spore coat protein around cadmium ions, the ions become isolated from the environment (and humans) and no longer have harmful effects. </li></ul><ul><li>Metallothionein = SmtA; Spore coat protein = CotC </li></ul><ul><li>NOVELTY – moving cadmium into resilient spores has not been accomplished before. </li></ul><ul><li>Future teams could apply same principle for other toxic metals. </li></ul>Cadmium Sequestration: What is this sub-project about?
    21. 22. Cadmium Sequestration: BioBrick Construct
    22. 23. Sporulation Tuning
    23. 24. Sporulation Tuning: Aims and Novelty <ul><li>Aim: </li></ul><ul><li>To adjust the natural sporulation system, instead of including something totally new. </li></ul><ul><li>Find out how the percentage of the population that sporulates, and does not sporulate, when the system is affected, by example, change in protein level. </li></ul><ul><li>Find the optimal percentage for the system in mind </li></ul><ul><li>Modelling: </li></ul><ul><li>Sin (sporulation inhibition) Operon Model (CellML) </li></ul><ul><li>Kin A Expression Model (COPASI) </li></ul><ul><li>Sporulation Induction using Kin A Model (COPASI) </li></ul>
    24. 25. Sporulation Tuning – the sporulation trigger <ul><li>The transcription factor Spo0A is a master regulator for entry into sporulation in the bacterium Bacillus subtilis [1] </li></ul><ul><li>Sporulation can be triggered with high efficiency in cells in the exponential phase of growth in rich medium by artificial induction of the synthesis of any one of three histidine kinases that feed phosphoryl groups into the relay [1] </li></ul><ul><li>For our project, we are using kinA as: </li></ul><ul><li>- In Bacillus subtilis , KinA is a major histidine kinase responsible for activation the sporulation pathway [2] </li></ul><ul><li>The KinA Expression Model illustrates how KinA is expressed while the third model shows how KinA induces sporulation </li></ul><ul><li>It is important to note, that a gradual increase in the Spo0A protein and activity plays a critical role in triggering sporulation and requires the action of the phosphorelay [1] </li></ul>
    25. 26. Sporulation Tuning: Modelling
    26. 27. Sporulation Tuning: Lab Work and Characterisation
    27. 28. References <ul><li>[1] Fujita, M. and Losick, R. 2005. Evidence that entry into sporulation in Bacillus subtilis is governed by a gradual increase in the level and activity of the master regulator Spo0A. Genes & Development 19: 2236-2244 </li></ul><ul><li>[2] Eswaramoorthy, P., Guo, T. and Fujita, M. 2009. In Vivo Domain-Based Functional Analysis of the Major Sporulation Sensor Kinase, Kin A, in Bacillus subtilis . Journal of Bacteriology p. 5358-5368 </li></ul>
    28. 29. Sin (sporulation inhibition) Operon Model <ul><li>Controls the production and activity of the repressor SinR </li></ul><ul><li>SinR in its active tetrametric form inhibits sporulation by repressing stage II and Spo0A promoters </li></ul><ul><li>Phosphorylation induces Spo0A to form active dimers which activate trascription from the P1 promoter </li></ul>
    29. 30. Population Modelling
    30. 31. Population Modelling: Aims and Novelty <ul><li>What is the affect of modifying the bacteria's life cycle? </li></ul><ul><ul><li>Does the population die, if we reduce germination? </li></ul></ul><ul><ul><li>How much of our bacteria will be needed to clean up an area? </li></ul></ul><ul><li>A shared environment. </li></ul><ul><li>Independent cells of bacteria making decisions for their life. </li></ul><ul><li>Each cell runs cellular models, using it's own parameters. </li></ul>
    31. 32. Population Modelling: How does it work? <ul><li>Programmed in Java, uses, Jsim, and other models written in CellML and SBML while integrating agent based models and biological models. </li></ul><ul><li>Due to each bacterial cell running independently as a thread, it used a lot of CPU power and RAM. </li></ul><ul><li>Powerful machines (16 CPUs) were used. </li></ul>
    32. 33. Population Modelling: Distributed Computing <ul><li>The solution – Distributed Computing – By using multiple computers to spread the load. </li></ul><ul><li>Using Microbase – It now runs on university computers and the Amazon Elastic Compute Cloud. </li></ul>Database Server (Microbase) ‏ Amazon EC2 Cell
    33. 34. Any Questions?