Team:Paris/Parts RBS
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- | 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 | + | 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. |
Revision as of 19:37, 21 October 2009
iGEM > Paris > Production > Parts Design
RBS
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