Lock/Key library: The plan

Lock construction (weak RBS (B0031) example)

1. Choose a RBS and make the complementary RNA strand

5’- UCACACAGGAAACC-3 RNA

2. Attach the nucleotide sequence UUUGGGUAGAUCAC upstream the RBS. This sequence will be part of the loop and contains a pyrimidine-uracil-nucleotide-purine (YUNR) consensus sequence that shown to be critical for the interaction key-lock.

5’- UUUGGGUAGAUCAC UCACACAGGAAACC- 3

3. (Optional) Attach the scar (UACUAG) and initiation codon (AUG) downstream the RBS

5’- UUUGGGUAGAUCAC UCACACAGGAAACC UACUAG AUG

4. Attach the nucleotide sequence GGAC upstream the last construct. This produces two mismatch nucleotides (red section underline).

5’- GGAC UUUGGGUAGAUCAC UCACACAGGAAACC UACUAG AUG – 3’

5. Attach the RBS’s* reverse complementary nucleotide sequence upstream the last construct.

5’- GGAUUCCUGUGUGA GGAC UUUGGGUAGAUCAC UCACACAGGAAACC UACUAG AUG – 3’

6. Attach the nucleotide sequence GUA, which is complementary to one part of the scar, upstream the last construct

5’- GUA GGAUUCCUGUGUGA GGAC UUUGGGUAGAUCAC UCACACAGGAAACC UACUAG AUG-3’

To sum-up, a simplified construction protocol is: add the RBS and the RBS’s reverse complementary* in the next sequence inside brackets 1 and 2 respectively.

5’- GUA [ 2 ] GGAC UUUGGGUAGAUCAC [ 1 ] UACUAG AUG-3’

In this step you have to change (arbitrarily) one nucleotide in the RBS for its complementary before to get the reverse complementary, this will give a mismatch. This mismatch is necessary in order to allow the key to easily open the lock.

Key construction (weak RBS (B0031) example)

1. Choose a RBS and make the complementary RNA strand.

5’- UCACACAGGAAACC-3 RNA

2. Attach the nucleotide sequence ACCCAAAGUCC upstream the RBS. This sequence is complementary to the loop formed in the lock.

5’- ACCCAAAGUCC UCACACAGGAAACC-3

3. Attach the sequence UGGUUAAUGAAAAUUAACUUA downstream the RBS. This sequence forms a loop and a helix with one mismatch.

5’- ACCCAAAGUCC UCACACAGGAAACC UGGUUAAUGAAAAUUAACUUA -3

4. Attach the RBS’s* reverse complementary nucleotide sequence downstream the last construct.

5’- ACCCAAAGUCC UCACACAGGAAACC UGGUUAAUGAAAAUUAACUUA GGUUUCCACUGUGA -3

5. (optional?) Attach the nucleotide sequence AAAAAGCCGAGUUAUUAAUCCGGCUU downstream the last construct. This sequence forms a second loop which may be useful for stability.

5’- ACCCAAAGUCC UCACACAGGAAACC UGGUUAAUGAAAAUUAACUUA GGUUUCCACUGUGA AAAAAGCCGAGUUAUUAAUCCGGCUU -3

To sum-up a simplified construction protocol is: add the RBS and the RBS’s reverse complementary* in the next sequence inside brackets 1 and 2 respectively.

5’-ACCCAAAGUCC [ 1 ] UGGUUAAUGAAAAUUAACUUA [ 2 ]AAAAAGCCGAGUUAUUAAUCCGGCUU -3

In this step you have to change (arbitrarily) two nucleotide in the RBS for its complementary before to get the reverse complementary, this will give a mismatched. This mismatch is necessary in order to allow the key to easily open the lock.

Furthermore, 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).

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 R0010 and E0240 in a Cloranphenicol biobrick backbone pSB1C3.

Figure 17. Control for lock/key for “weak” ribosome binding site. The construction was made using the Biobricks R0010, B0031 and J04630 in a Cloranphenicol biobrick backbone pSB1C3.

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