Team:TUDelft/New Lock Key Biobricks

=New Biobricks Design=

We want to test if the algorithm delivers functional locks and keys. In order to do that, we plan to characterize two locks and keys generated by our algorithm. The RBS’s chosen were the weak and medium (biobricks) from the registry. The DNA sequences of these new parts are:

Lock for weak RBS (92 nt)

5´- GAATTC GCGGCCGC T TCTAGA G GTA GGATTCCTGTGTGA  GGAC TTTGGGTAGATCAC  TCACACAGGAAACC T ACTAGT A GCGGCCG CTGCAG -3´

3´- CTTAAG CGCCGGCG  A  AGATCT  C  CAT  CCTAAGGACACACT  CCTG  AAACCCATCTAGTG  AGTGTGTCCTTTGG  A  TGATCA  T  CGCCGGC  GACGTC- 5´

Key for weak RBS (129 nt)

5´-GAATTC GCGGCCGC T TCTAGA G ACCCAAAGTCC TCACACAGGAAACC TGGTTAATGAAAATTAACTTA GGTTTCCACTGTGA AAAAAGCCGAGTTATTAATCCGGCTT T ACTAGT A GCGGCCG CTGCAG-3´

3´- CTTAAG CGCCGGCG A AGATCT C TGGGTTTCAGG AGTGTGTCCTTTGG ACCAATTACTTTTAATTGAAT CCAAAGGTGACACT   TTTTTCGGCTCAATAATTAGGCCGAA A  TGATCA  T  CGCCGGC  GACGTC- 5´

Lock for medium RBS (90 nt)

5´- GAATTC GCGGCCGC T TCTAGA G GTA CTATCCTGTGTGA GGAC TTTGGGTAGATCAC TCACACAGGAAAG T ACTAGT A GCGGCCG CTGCAG-3´

3´- CTTAAG CGCCGGCG  A  AGATCT  C CAT  GATAGGACACACT  CCTG  AAACCCATCTAGTG  AGTGTGTCCTTTC A  TGATCA  T  CGCCGGC  GACGTC- 5´

Key for medium RBS (127 nt)

5´- GAATTC GCGGCCGC T TCTAGA G ACCCAAAGTCC TCACACAGGAAAG TGGTTAATGAAAATTAACTTA CTTTCCTGACTGA AAAAAGCCGAGTTATTAATCCGGCTT T ACTAGT A GCGGCCG CTGCAG -3´

3´- CTTAAG CGCCGGCG  A  AGATCT  C  TGGGTTTCAGG  AGTGTGTCCTTTC  ACCAATTACTTTTAATTGAAT  GAAAGGACTGACT  TTTTTCGGCTCAATAATTAGGCCGAA  A  TGATCA  T  CGCCGGC  GACGTC- 5´

These sequences will be incorporated as new biobricks to the registry and will be used to construct the next assemblies (figures 14 and 15).

CAUTION!!!, the previous sequences are intended to generate a RNA secondary structure after transcription. In DNA synthesis, a similar structure may be formed causing problems. To overcome this problem we, with the recommendations of some members of Berkeley iGEM 2006, Caltech iGEM 2007 and Peking iGEM 2007, developed a basic protocol for Lock/key synthesis.





In order to obtain confident data, we also design control biobricks. The construction of these controls is shown in figure 16 and figure 17.



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