Team:Paris/Parts RBS

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RBS

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.

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