Lock construction (weak RBS ([http://partsregistry.org/Part:BBa_B0031 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 ([http://partsregistry.org/Part:BBa_B0031 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.

The result of this algorithm can be found in the Lock/Key Generator

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