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 help of some members of , developed a basic protocol for Lock/key synthesis.

Figure 14. Characterization of lock and key for medium RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]). Before IPTG induction, GFP will be transcribed but due to the lock the translation will be blocked. After IPTG addition, the production of the key will start and the expression of GFP will lead to fluorescence. The construction was made using the Biobricks [http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_R0040 R0040], [http://partsregistry.org/Part:BBa_J04630 J04630] and the new biobricks designed here (LM: lock for medium RBS and KM: key for LM) in a Cloranphenicol biobrick backbone [http://partsregistry.org/Part:pSB1C3 pSB1C3].

Figure 15. Characterization of lock and key for weak RBS ([http://partsregistry.org/Part:BBa_B0031 B0031]). Before IPTG induction, GFP will be transcribed but due to the lock the translation will be blocked. After IPTG addition, the production of the key will start and the expression of GFP will lead to fluorescence. The construction was made using the Biobricks [http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_B0015 B0015], [http://partsregistry.org/Part:BBa_R0040 R0040], [http://partsregistry.org/Part:BBa_J04630 J04630] and the new biobricks designed here (LW: lock for medium RBS and KW: key for LW) in a Cloranphenicol biobrick backbone [http://partsregistry.org/Part:pSB1C3 pSB1C3].

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

Figure 16. Control for lock/key for “medium” ribosome binding site. The construction was made using the Biobricks [http://partsregistry.org/Part:BBa_R0010 R0010] and [http://partsregistry.org/Part:BBa_E0240 E0240] in a Cloranphenicol biobrick backbone [http://partsregistry.org/Part:pSB1C3 pSB1C3].

Figure 17. Control for lock/key for “weak” ribosome binding site. The construction was made using the Biobricks [http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_B0031 B0031] and [http://partsregistry.org/Part:BBa_J04630 J04630] in a Cloranphenicol biobrick backbone [http://partsregistry.org/Part:pSB1C3 pSB1C3].

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