IGEM_Presentation_to_ChELSI_24th_Oct.ppt - Page - 2008.igem.org - iGEM

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IGEM_Presentation_to_ChELSI_24th_Oct.ppt - Page - 2008.igem.org -  iGEM Powered By Docstoc
					International Genetically
  Engineered Machines
      Competition
          M.I.T, Nov 7th-9th
          2008




 An introduction to the University of
   Sheffield 2008 iGEM Team…
                        University Of Sheffield 2008 iGEM Team




What is iGEM?
   iGEM is a rapidly increasing international
    competition for undergraduates in many different
    specialisations
    –   Designed to involve undergraduates in research early in their
        careers
    –   Over 84 teams from all around the world this year

   Premise is to expand on the principle of synthetic
    biology
    –   Pieces of DNA are designed and standardised at each end, in the
        hope of building novel organisms
    –   Information made publicly available
    –   ‘Wiki’
                                                                   Summer 2008
                University Of Sheffield 2008 iGEM Team




Who are we?
     Gosia Poczopko                   Dmitry Malyshev
     1st year Molecular and                1st year Biomedical
     Cellular Biochemist                              Engineer




     Eva Barkauskaite                    Hammad Karim
     1st year Biochemist                     2nd year Engineer




     Rosie Bavage
                                          Sam Awotunde
     1styear Molecular
     Biologist                                 2nd year Engineer




                                                                   Summer 2008
              University Of Sheffield 2008 iGEM Team




The Idea
 A biosensor for cholera in drinking water –
  machine/test/kit
 We want to hijack a pathway in E.coli and
  manipulate it to detect Vibrio cholerae
  quorum sensing autoinducers
 GFP marker inserted downstream
 Proof of principle in fusion kinase



                                                       Summer 2008
        University Of Sheffield 2008 iGEM Team




BarA Pathway

                                                 • More than 20 target
                                                 genes for UvrY

                                                 • Includes glycogen
                                                 synthesis, glycolysis,
                                                 gluconeogenesis,
                                                 glycogen catabolism.

                                                 • Our target: PGA
                                                 operon – role in
                                                 biofilm formation


                                                                  Summer 2008
                   University Of Sheffield 2008 iGEM Team




Fusion Receptor
   Expression of membrane bound
    receptor sensing V. cholera
    signalling molecule in E.coli
   Novel approach – to fuse receiver
    and transmitter domain from two
    related receptors
   Receptors are closely related and
    have similar topology
   Fused receptor:
          CqsS – V.cholerae


          BarA – E.coli



                                                            Summer 2008
                      University Of Sheffield 2008 iGEM Team




Fusion Receptor
   Sequences to be fused were found through multisequene allignment
    and comparison with similar proteins




                                                               Summer 2008
                       University Of Sheffield 2008 iGEM Team




Fusion Receptor
   Pathways regulated via BarA are well characterised




                                                                Summer 2008
            University Of Sheffield 2008 iGEM Team




GFP into genome
 GFP will act as our reporter
 Inserted into the genome under the
  promoter of PGA operon between PGAa
  and PGAb




                                                     Summer 2008
             University Of Sheffield 2008 iGEM Team




Gene Knockout
 To make sure native BarA doesn’t trigger
  the production of GFP, we need to knock
  out certain genes from our strain
 Using Datsenko and Wanner’s method for
  speeding up recombination
 PCR products provide homology, λ Red
  recombinase system provides faster
  recombination.
 Marker gene removed later

                                                      Summer 2008
        University Of Sheffield 2008 iGEM Team




Gene Knockout




                                                 Summer 2008
                     University Of Sheffield 2008 iGEM Team




Problems
   We couldn’t get a knockout
    –   5 repeats, with varied condition
   Various setbacks and little time
    –   Ampicillin




                                                              Summer 2008
              University Of Sheffield 2008 iGEM Team




CAI-1 Synthesis
 CqsA is the synthesis machine for CAI-1’s
  in cholera
 Bonnie Basslers lab designed plasmid and
  protocol for transferring CqsA into E.coli
  and purify the CAI-1 product – it works
 Received and used
 Mass-spec to confirm been difficult to obtain



                                                       Summer 2008
                     University Of Sheffield 2008 iGEM Team




Further ideas
   Re-usuable sensor
    –   Cleavable GFP/ housekeeping gene regulation – LVA
        tag.
    –   Provided by past iGEM project = criteria for an award
   Threshold experiments
    –   Modelled




                                                              Summer 2008
BioBrick - Characterization
 Plan: Insertion of GFP-LVA under pgaABCD
  operon.
Why?
 Reporter
 GFP-LVA gene previous BioBrick = Criteria for
  ‘Silver Award’
 LVA tag attracts housekeeping protease –
  degradation/reusable
 Lac promoter = inducible, for measurement of
  fluorescence
What has been done?
 DH5-alpha transformed with an
  uncharacterized GFP-LVA BioBrick
 Used Tecan® , with fluorescence
  measurements every 15 minutes for 8
  hours
Results 1
 5 repeated measurements, with consistent
  lack of fluorescence
 Tried RFP-LVA (uncharacterized but made
  by different team) and characterized, tested
  RFP
 Transformation 1 failed, transformation 2 in
  MBB failed despite successful positive
  controls
Conclusion
 Not one successful transformation, despite
  using tested BioBricks
 A lot of troubleshooting, from various
  advisors
 Last attempt: carried out by PhD student,
  which failed
 Conclusion: BioBrick booklet may have
  been faulty. However has not been proven.
Heath and Safety
 Vibrio cholerae impossible to work on
 CAI-1s non-toxic themselves
 Repress Cholerae biofilm formation in
  nature
 CqsA only produces CAI-1s
 Safe
                       University Of Sheffield 2008 iGEM Team




Acheive: Bronze Award
   Register
   Complete and submit a Project Summary form.
   Create an iGEM wiki
   Present a Poster and Talk at the iGEM Jamboree
   Enter information detailing at least one new standard BioBrick Part or
    Device in the Registry of Parts
     – including nucleic acid sequence, description of function,
       authorship, safety notes, and sources/references.
   Submit DNA for at least one new BioBrick Part or Device to the
    Registry of Parts
   We’ve done all of these

                                                                   Summer 2008
                 University Of Sheffield 2008 iGEM Team




Engineering - Sam
 Synthetic biology is the application of
  engineering principles and approach to
  molecular biology
 Mathematical modelling of our BarA/UvrY
  system , with fluorescence of GFP, allows
  its dynamics and behaviour to be analysed
 The model is validated in a two steps:
    •   The signal transduction
    •   The gene expression.

                                                          Summer 2008
A Two-component Signal Transduction System

 Sensor kinase                Response regulator    Uvry.DNA




      BarA~p
                                      Uvry~p
                                                      R4




       R1         R2                   R3              DNAf




                                                    DNA binding
                                       Uvry
      BarA

                 Phosphoryl
                 transfer       dephosphorylation
The Chemical Reactions
•  Auto-phosphorylation:
ATP + BarA ↔ ADP + BarA~p --Reaction 1
•   Phosphoryl group transfer :
BarA~p + UvrY ↔ BarA + UvrY~p - Reaction 2
•   Dephosphorylation :
  UvrY~p + BarA → UvrY + BarA (+ pi) -Reaction
   3
• DNA binding :
2 UvrY~p + DNAƒ ↔ (UvrY – DNA)----Reaction 4
Reaction Analyses
• In reaction R1, the stimulus enhances the kinase activity that
  results in auto-phosphorylation of sensor kinase (BarA, BarA~p
  state variable) by ATP
• In reaction R2 the phosphoryl group is transferred to th response
  regulator (Uvry, Uvry~p state variable). Uvry~p contains the active
  output domain.
• Reaction R3 describes the dephosphorylation of Uvry~p by
  cognate sensor kinase BarA. (it has been shown through reference
  that dephosphorylation is only dependent on BarA. Jung et.al.,
  1997) so that other phosphatises are not considered in the model.
• In reaction R4 the activated response regulator forms a dimer and
  is then binds to the free DNA (DNAf, state variable) to build a
  transcription complex (Uvry-DNA, state variable), in presence of
  RNA polymerase.
Differential Equations
Phosphorylation Rate of
BarA
Phosphorylation Rate of
UvrY
Rate of GFP Gene
Expression
Parameter Values
 In vitro parameters
 K1 = o.oo29 1/h µM    DNA₀ = 100µM

 k_1= 0.00088 1/hµM    BarA₀ = 1µM

 K2= 108 1/hµM          Uvry₀ = 4µM

 K_2= 1080 1/hµM       ATP = 100µM

 Kь = 5400 1/hµM       ADP = 8µM

 K_ь = 360 1/h

 K3= 90 1/hµM
Conclusions
•   The model and simulation was carried-out
    in Matlab and the dynamics of the system
    was studied.
•   The parameters with highest sensitivity
    were k1, kb, k3, k_b.
•   The response of the autophosphorylation
    and phosphorylation of the BarA, Uvry and
    the expression of the gene respectively
    show that the system is stable and under
    any conditions it should respond well.
                    University Of Sheffield 2008 iGEM Team




Engineering –                        Hammad’s Probabilistic approach

   For simplicity, whole reaction is split into
    two parts:
    –   CAI-1 interacting with Fusion Kinase
    –   From response regulatory protein to GFP glow.
   Mathematically,
    –




                                                             Summer 2008
                         University Of Sheffield 2008 iGEM Team




Engineering – Hammad’s Probabilistic approach
   Considering this as Poisson Process:
    –   The General form of probability is then given as:




    –   Also this interaction will follow law of diffusion (ideal case), thus
        probability of reaction rate increasing with time can be given as
        Gaussian distribution :




                                                                        Summer 2008
                       University Of Sheffield 2008 iGEM Team




Engineering - The Probabilistic approach

   Implementation:
•   As there are some other processes occurring at the same time (like
    noise disturbance and various reactions) using Gaussian mixture
    models :




                                                                 Summer 2008
                 University Of Sheffield 2008 iGEM Team




Engineering - The Probabilistic approach




      Probability curves of contact between molecules


                                                          Summer 2008
             University Of Sheffield 2008 iGEM Team




Sponsors
 idtDNA – £1000 gene, and 10 free primers
 iChemE - £1000 reimbursement for travel
 £2500 from Prof Poole MBB (covered all
  flights and hotels)
 Printing and other minor costs from MBB
  Funds



                                                      Summer 2008
                        University Of Sheffield 2008 iGEM Team




Our many thanks go to…
   Prof Philip Wright
   Dr Catherine Biggs
   Esther Karunakaran
   other ChELSI members
   Dave Wengraff
   Prof David Hornby
   Prof Robert Poole
   Prof Visakan Kadirkhamanatan
   Prof David Rice
   Prof Jeff Green
   The Bassler, Stafford and Karolinska Institute labs for plasmid
    provision.                                                        Summer 2008
                            University Of Sheffield 2008 iGEM Team




References
   Datsenko & Wanner, 2000, ‘One-step inactivation of chromosomal genes in Escherichia
    coli K-12 using PCR products’
   Higgins, Bassler et al, 2007, ‘The major Vibrio cholerae autoinducer and its role in
    virulence factor production’
   Hammer & Bassler, 2007, ‘Regulatory small RNAs circumvent the conventional quorum
    sensing pathway in pandemic Vibrio cholerae’
   Jun Zhu, Melissa B. Miller, et al, 2001, ‘Quorum-sensing regulators control virulence
    gene expression in Vibrio cholerae’
   Tomenius, Pernestig et al, 2005, ‘Genetic and functional characterization of the E.coli
    BarA-UvrY Two-componant system’
   Suzuki et al, 2002, ‘Regulatory Circuitry of thr CsrA/CrsB and BarA/UvrY systems of
    E.coli’
   Sahu, Acharya et al, 2003, ‘The bacterial adaptive response gene, barA, encodes a
    novel conserved histidine kinase regulatory switch for adaptation and modulation of
    metabolism in E.coli
   Andersen, J.B et al. 1998, ‘New Unstable Variants of Green Fluorescent Protein for
    Studies of Transient Gene Expression in Bacteria’

                                                                                 Summer 2008

				
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