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                      Outer Membrane Vesicles (OMVs)

                                                     • Derived from gram-negative
                                                       bacterial outer membrane
                                                     • 20-200nm in diameter
                                                     • Carry biological molecules




                  DNA                                Signalling molecules
                                                  zycnzj.com/http://www.zycnzj.com/                   Proteins
  2009 DeLisa et al: Demonstrate co-localisation of proteins to OMVs using heterologous
  protein fusions with ClyA toxin
Picture adapted from McBroom et al. Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response,
Molecular Microbiology 63(2), 545-558
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    Engineering advantages




 Protection
                       Harvest
                 zycnzj.com/http://www.zycnzj.com/   Communication
    from
degradation
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VESECURE                               FOUNDATIONAL TOOLS



    Wetlab                                      Bioscaffold




   Modelling                                     BSim
            zycnzj.com/http://www.zycnzj.com/
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      Series switch




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                       n
 number of toggle switches in the series switch




n=2                                            n=3
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                                   Δ
                     time before switches pop open




           Δ→0                                               Δ→∞
(switches pop open straight away)                  (switches stay shut indefinitely)
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     Packaging method
             packaged or unpackaged




Packaged                                         Unpackaged
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                       Is packaged delivery beneficial?



                       n           number of toggle switches in the series switch
Proportion activated




                       Δ           time before switches pop open



                       Packaging method
                                   zycnzj.com/http://www.zycnzj.com/
                                                                       packaged or unpackaged




                            Time
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                                 Results for Δ → 0
                                    (switches pop open straight away)

                        1


                       0.8
Proportion activated




                                                             packaged
                       0.6


                       0.4
                                                             unpackaged, n = 2
                       0.2
                                         zycnzj.com/http://www.zycnzj.com/


                        0
                         0   20          40            60
                             Time / seconds
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                                 Results for Δ → ∞
                                      (switches stay shut indefinitely)

                        1                                   unpackaged, n = 2
                                                            unpackaged, n = 3
                       0.8                                  unpackaged, n = 4
Proportion activated




                                                            packaged
                                                            unpackaged, n = 5
                       0.6


                       0.4


                       0.2
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                        0
                         0   20          40            60
                             Time / seconds
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VESECURE                               FOUNDATIONAL TOOLS



    Wetlab                                     Bioscaffold




   Modelling                                     BSim
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             Biobrick structure


Promoter       RBS       Carrier         Linker          Cargo   Terminator




AraC        J61100      FhuA                             GFP      B0014
                        OsmE
                          Fiu


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                  Biobricks submitted

Promoter    RBS                    AraC-RBS
                                    (K259007)

  Carrier                          FhuA            OsmE
                                (K259000)        (K259011)

Promoter    RBS     Carrier         AraC-RBS-FhuA                 AraC-RBS-OsmE
                                        (K259010)                    (K259012)

  Cargo      Terminator             GFP-Terminator
                                        (K259006)
                              zycnzj.com/http://www.zycnzj.com/
 Finished assemblies                AraC-RBS-FhuA-GFP             AraC-RBS-GFP-Terminator
                                        (K259013)                     (K259014)
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                        Assessment of carrier proteins
                                Promoter Induction
                     0.6
                                                                             GFP
                     0.4                                                     FhuA
log (10) O.D. A600




                     0.2
                       0                                                              Time (min)
                     -0.2 0      50     100        150        200           250     300

                     -0.4                                                     FhuA-GFP
                     -0.6                                                     OsmE
                     -0.8               zycnzj.com/http://www.zycnzj.com/


                      -1
                     -1.2
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  VESECURE summary


                Showed it can be beneficial to package
Modelling
                proteins into vesicles




             Determined certain carriers might be
             inappropriate for
 Wetlab zycnzj.com/http://www.zycnzj.com/ protein fusions
              Submitted 13 Biobricks
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VESECURE                               FOUNDATIONAL TOOLS



    Wetlab                                     Bioscaffold




   Modelling                                     BSim
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               Protein fusions

                           Remove
Carrier                     Scars
                                                            DNA
                                                  Carrier   SCAR   Cargo

                Remove
Cargo            Stop
                Codons
                                      Maintain
                                     proteins in
                                       frame
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                Current assembly standards

                     RFC 25                      RFC 23                   RFC 15
             Modifies prefix/suffix to    Modifies prefix/suffix     Removes scar and
Solution :
              accomodate fusion          DNA scar does not give        stop codons
                                              stop codon
Problem :     Parts must take into       Arginine residue in scar   Used on case-by-case
             account extra R.E. sites                                      basis



                    Neither upwards compatible with RFC10
                                zycnzj.com/http://www.zycnzj.com/
                (stipulates a 3’ TAA TAA to protein coding parts)
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                      Bioscaffold (RFC44)

         BpuEI      BpuEI           Tyr                  Met          BseRI   BseRI


5’-                                                                                   -3’
      KEY:

      BpuEI        Restriction Enzyme Sequence and Orientation
                   Translational Buffer
             Tyr   Aminoacid      zycnzj.com/http://www.zycnzj.com/


                   Gly-Ser-Gly Bridge
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     Bioscaffold application

NNNTA ATAATACTAGAG(N)6CTCGGC(N)6GCCGAG(N)18 TA CGGCAGCGGTATG




NNNTACGGCAGCGGTATG(N)10CTCCTC(N)12GAGGAG(N)2TACTAGATGNNN
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VESECURE                               FOUNDATIONAL TOOLS



    Wetlab                                     Bioscaffold




  Modelling                                      BSim
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  Modelling in synthetic biology




                                                   ?
Intracellular                             Multicellular
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              BSim then and now
A software package for realistic simulation of multicellular phenomena




                        zycnzj.com/http://www.zycnzj.com/




                                 2009
                                 2008
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                       New features



Force-based      Growth and            Diffusive             Brownian             Custom
 dynamics        replication          chemicals               motion            interactions




                          zycnzj.com/http://www.zycnzj.com/
 Vesiculation       Custom            Delivery            Accurate model          Custom
                   behaviour                              of E. coli motility     output
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                  Multicellular and multilevel


                                                              2008   2009



 Intracellular dynamics




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Multicellular phenomena
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       Case studies




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               Synchronization ofof in swarm immunity
                      comparison coupled genetic and chemotaxis
                          of vesicles magnetotaxis
         QuantitativeRole Paris vesiculation networkoscillators
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             Getting BSim



http://code.google.com/p/bsim-bccs/


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Foundational advance summary


                    Developed a Biobrick friendly protein
Bioscaffold         fusion method that supports RFC10
                    (4 Biobricks)




                   Multicellular and multilevel modelling
 BSim      zycnzj.com/http://www.zycnzj.com/
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                              Thank you


Instructors & Advisors:
Mario di Bernardo
Caroline Colijin
Krasimira Tsaneva Atanasova
John Hogan
Claire Grierson
Nigel Savery
Athanasios Polynikis
Thomas Gorochowski            zycnzj.com/http://www.zycnzj.com/
Oliver Purcell

				
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