Team:Paris/Production design

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Parts used for the production of vesicles

Our contributions

  • TolRII
  • Colicin E3
  • etc...

NB: All coding sequences intended to be expressed are associated with a standard RBS incorporated in the forward primer used to amplify the sequence by PCR. The standard RBS used was carefully designed to ensure maximal functionality (see <a href="https://2009.igem.org/Team:Paris/Parts_RBS#bottom"> RBS</a>)


The RBS were designed by Guillaume Cambray and integrated directly inside the forward primer to yield expressible units.


The RBS sequence used is the following: 5'-AAGGAGGTATATA-3', immediately followed by the ATG initiation codon.


The AAGGAGG motif is complementary to the 16S RNA of the ribosome and ensure full recognition by the translational machinery. The first A overlap the XbaI site to reduce primer length. The distance between the first GG and the initiation codon is set to 9 bp, which appear to be the optimal spacing according to Anatomy of Escherichia coli ribosome binding sites - Shultzaberger et al, 2001. The (TA) repeats spacer is used because the GC content of the RBS should be low to favor the separation of the DNA strand. Alternating T and A are meant to avoid errors during primer synthesis. At last, this spacer is designed so that the -3 bp before ATG is an adenine, which may be important according to old reports.


Overall the forward primer used for amplifying CDS has the following template sequence: 

5'-TTGAATTCGCGGCCGCTTCTAGAAGGAGGTATATAATG-3'
   -- 2 bases to ensure efficient EcoRI cutting while minimizing primer length
     --------------------- Biobrick standard RFC10
                         ------- RBS
                                ------ Low GC spacer
                                   - potentially important A
                                      --- Start codon

Registered parts we used

Backbones

The function "increased production of OMV" is borne on two plasmid backbones [http://partsregistry.org/Part:pSB1A3 pSB1A3] and [http://partsregistry.org/Part:pSB3T5 pSB3T5].

Biobricks

  • Expression of the proteins targeted to OMVs as well as lacI is controled by the Pbad promoter [http://partsregistry.org/Part:BBa_K113009 BBa_K11309].

We used the Ptet promoter [http://partsregistry.org/Part:BBa_R0040 BBa_ROO40] and the coding sequence for the TetR repressor protein [http://partsregistry.org/Part:BBa_C0040 BBa_COO40] to tightly repress the expression of protein domains that are expected to destabilize the outer membrane, thereby leading to increased production of OMVs. We hope that TetR mediated repression is strong enough to avoid leakage and weak expression of these deleterious polypeptides.

  • The Plac promoter [http://partsregistry.org/Part:BBa_I14032 BBa_I14032] associated to lacI [BBa_C0012 http://partsregistry.org/Part:BBa_C0012] to control the expression of tetR.
  • The double terminator [http://partsregistry.org/Part:BBa_B0014 BBa_B0014] was used to isolates the different expression units from each other.

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