"
Team:TUDelft/Algorithm
From 2009.igem.org
(→Key construction (weak RBS (B0031) example)) |
Danielsoes (Talk | contribs) |
||
| (2 intermediate revisions not shown) | |||
| Line 1: | Line 1: | ||
{{Template:TUDelftiGEM2009}} | {{Template:TUDelftiGEM2009}} | ||
| - | |||
| - | |||
| - | |||
==Lock construction (weak RBS ([http://partsregistry.org/Part:BBa_B0031 B0031]) example)== | ==Lock construction (weak RBS ([http://partsregistry.org/Part:BBa_B0031 B0031]) example)== | ||
1. Choose a RBS and make the complementary RNA strand | 1. Choose a RBS and make the complementary RNA strand | ||
| - | '''5’- UCACACAGGAAACC- | + | '''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. | 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- | + | '''5’- UUUGGGUAGAUCAC UCACACAGGAAACC- 3’''' |
3. (Optional) Attach the scar (UACUAG) and initiation codon (AUG) downstream the RBS | 3. (Optional) Attach the scar (UACUAG) and initiation codon (AUG) downstream the RBS | ||
| - | '''5’- UUUGGGUAGAUCAC UCACACAGGAAACC UACUAG AUG''' | + | '''5’- UUUGGGUAGAUCAC UCACACAGGAAACC UACUAG AUG -3’''' |
4. Attach the nucleotide sequence GGAC upstream the last construct. This produces two mismatch nucleotides (red section underline). | 4. Attach the nucleotide sequence GGAC upstream the last construct. This produces two mismatch nucleotides (red section underline). | ||
| Line 39: | Line 36: | ||
1. Choose a RBS and make the complementary RNA strand. | 1. Choose a RBS and make the complementary RNA strand. | ||
| - | '''5’- UCACACAGGAAACC- | + | '''5’- UCACACAGGAAACC-3’ RNA''' |
2. Attach the nucleotide sequence ACCCAAAGUCC upstream the RBS. This sequence is complementary to the loop formed in the lock. | 2. Attach the nucleotide sequence ACCCAAAGUCC upstream the RBS. This sequence is complementary to the loop formed in the lock. | ||
Latest revision as of 14:18, 8 October 2009
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 -3’
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
