Team:TUDelft/Algorithm

From 2009.igem.org

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(New page: ''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...)
 
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''Lock construction (weak RBS ([http://partsregistry.org/Part:BBa_B0031 B0031]) example)''
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{{Template:TUDelftiGEM2009}}
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==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
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5’- UCACACAGGAAACC-3 RNA
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'''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.
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5’- UUUGGGUAGAUCAC  UCACACAGGAAACC- 3
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'''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
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5’- UUUGGGUAGAUCAC  UCACACAGGAAACC  UACUAG  AUG
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'''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).
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5’- GGAC UUUGGGUAGAUCAC  UCACACAGGAAACC  UACUAG  AUG – 3’
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'''5’- GGAC UUUGGGUAGAUCAC  UCACACAGGAAACC  UACUAG  AUG – 3’'''
5. Attach the RBS’s* reverse complementary nucleotide sequence upstream the last construct.
5. Attach the RBS’s* reverse complementary nucleotide sequence upstream the last construct.
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5’- GGAUUCCUGUGUGA  GGAC UUUGGGUAGAUCAC  UCACACAGGAAACC  UACUAG  AUG – 3’
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'''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
6. Attach the nucleotide sequence GUA, which is complementary to one part of the scar, upstream the last construct
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5’- GUA  GGAUUCCUGUGUGA  GGAC UUUGGGUAGAUCAC  UCACACAGGAAACC  UACUAG AUG-3’
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'''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.
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.
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5’- GUA  [ 2 ]  GGAC UUUGGGUAGAUCAC  [ 1 ]  UACUAG AUG-3’
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'''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.
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.
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''Key construction (weak RBS (B[http://partsregistry.org/Part:BBa_B0031 B0031]) example)''
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==Key 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.
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5’- UCACACAGGAAACC-3                RNA
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'''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.
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5’- ACCCAAAGUCC  UCACACAGGAAACC-3
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'''5’- ACCCAAAGUCC  UCACACAGGAAACC-3’'''
3. Attach the sequence UGGUUAAUGAAAAUUAACUUA downstream the RBS. This sequence forms a loop and a helix with one mismatch.
3. Attach the sequence UGGUUAAUGAAAAUUAACUUA downstream the RBS. This sequence forms a loop and a helix with one mismatch.
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5’- ACCCAAAGUCC  UCACACAGGAAACC  UGGUUAAUGAAAAUUAACUUA -3
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'''5’- ACCCAAAGUCC  UCACACAGGAAACC  UGGUUAAUGAAAAUUAACUUA -3’'''
4. Attach the RBS’s* reverse complementary nucleotide sequence downstream the last construct.
4. Attach the RBS’s* reverse complementary nucleotide sequence downstream the last construct.
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5’- ACCCAAAGUCC  UCACACAGGAAACC  UGGUUAAUGAAAAUUAACUUA  GGUUUCCACUGUGA -3
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'''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. (optional?) Attach the nucleotide sequence AAAAAGCCGAGUUAUUAAUCCGGCUU downstream the last construct. This sequence forms a second loop which may be useful for stability.
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5’- ACCCAAAGUCC  UCACACAGGAAACC  UGGUUAAUGAAAAUUAACUUA  GGUUUCCACUGUGA  AAAAAGCCGAGUUAUUAAUCCGGCUU -3
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'''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.
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.
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5’-ACCCAAAGUCC [ 1 ] UGGUUAAUGAAAAUUAACUUA [ 2 ]AAAAAGCCGAGUUAUUAAUCCGGCUU -3
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'''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.
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.
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The result of this algorithm can be found in the [https://2009.igem.org/Team:TUDelft/RiboKeyLock_Generator Lock/Key Generator]
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[https://2009.igem.org/Team:TUDelft/Lock/Key_library Return]
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<br>
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{{Template:TUDelftiGEM2009_end}}

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

Return