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Team:TUDelft/Algorithm
From 2009.igem.org
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 (BB0031) 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.
