Difference between revisions of "Part:BBa J119367"
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tClone is a construction intermediate that can be used to build devices that will allow users to clone and test new transcriptional terminators and riboswitches that function by antitermination without gel purification or other preparation of DNA. Use of BioBrick cloning to add the RFP gene into the suffix of tClone produce tClone Red, which can be used as a reporter for cloning and testing new transcriptional terminators and riboswitches. Similarly, use of BioBrick cloning to insert the TetA gene into the suffix of tClone produces tClone TetA, which can be used to measure new transcriptional terminators and riboswitches. tClone vectors can be used as destination vectors for Golden Gate Assembly (GGA) using BsaI and ligase. A new terminator or riboswitch can be derived from synthetic oligos, PCR, or a plasmid clone. For proper assembly, the new insert must have the appropriate 4 nt sticky ends or be flanked by BsaI sites that produce the sticky ends. With reference to the top strand, the left site must be 5' CGAC 3' and the right site must be 5' GCGG 3'. BsaI always produces a 5' overhang sticky end. Successful GGA assembly replaces the reverse promoter driving GFP expression with the new terminator or riboswitch. Transcription will be initiated in the direction of the RFP coding sequence. Whether or not transcription proceeds to the RFP gene is determined by the strength of the terminator or whether termination or antitermination occurs at the riboswitch. The part incorporates the BD18 bicistronic translational junction (see Part:BBa_J119024) engineered by Vivek Mutalik and The BIOFAB Team at biofab.org. | tClone is a construction intermediate that can be used to build devices that will allow users to clone and test new transcriptional terminators and riboswitches that function by antitermination without gel purification or other preparation of DNA. Use of BioBrick cloning to add the RFP gene into the suffix of tClone produce tClone Red, which can be used as a reporter for cloning and testing new transcriptional terminators and riboswitches. Similarly, use of BioBrick cloning to insert the TetA gene into the suffix of tClone produces tClone TetA, which can be used to measure new transcriptional terminators and riboswitches. tClone vectors can be used as destination vectors for Golden Gate Assembly (GGA) using BsaI and ligase. A new terminator or riboswitch can be derived from synthetic oligos, PCR, or a plasmid clone. For proper assembly, the new insert must have the appropriate 4 nt sticky ends or be flanked by BsaI sites that produce the sticky ends. With reference to the top strand, the left site must be 5' CGAC 3' and the right site must be 5' GCGG 3'. BsaI always produces a 5' overhang sticky end. Successful GGA assembly replaces the reverse promoter driving GFP expression with the new terminator or riboswitch. Transcription will be initiated in the direction of the RFP coding sequence. Whether or not transcription proceeds to the RFP gene is determined by the strength of the terminator or whether termination or antitermination occurs at the riboswitch. The part incorporates the BD18 bicistronic translational junction (see Part:BBa_J119024) engineered by Vivek Mutalik and The BIOFAB Team at biofab.org. | ||
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| − | NOTE: this version was later | + | NOTE: this version was later tested with riboswitch PDB [[Part:BBa_J100255]] (containing promoter P5 and RBS BD18) which was too leaky to be useful. Therefore, it might be best to use promoter P9 and BD10 instead in subsequent applications of tClone Tet. See [[Part:BBa_J100279]] and its experience page. |
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| − | [[File: | + | [[File:tClone.png]] |
</center> | </center> | ||
Latest revision as of 13:45, 28 September 2016
tClone: Device for Testing Transciptional Terminators and Riboswitches via Golden Gate Assembly
tClone is a construction intermediate that can be used to build devices that will allow users to clone and test new transcriptional terminators and riboswitches that function by antitermination without gel purification or other preparation of DNA. Use of BioBrick cloning to add the RFP gene into the suffix of tClone produce tClone Red, which can be used as a reporter for cloning and testing new transcriptional terminators and riboswitches. Similarly, use of BioBrick cloning to insert the TetA gene into the suffix of tClone produces tClone TetA, which can be used to measure new transcriptional terminators and riboswitches. tClone vectors can be used as destination vectors for Golden Gate Assembly (GGA) using BsaI and ligase. A new terminator or riboswitch can be derived from synthetic oligos, PCR, or a plasmid clone. For proper assembly, the new insert must have the appropriate 4 nt sticky ends or be flanked by BsaI sites that produce the sticky ends. With reference to the top strand, the left site must be 5' CGAC 3' and the right site must be 5' GCGG 3'. BsaI always produces a 5' overhang sticky end. Successful GGA assembly replaces the reverse promoter driving GFP expression with the new terminator or riboswitch. Transcription will be initiated in the direction of the RFP coding sequence. Whether or not transcription proceeds to the RFP gene is determined by the strength of the terminator or whether termination or antitermination occurs at the riboswitch. The part incorporates the BD18 bicistronic translational junction (see Part:BBa_J119024) engineered by Vivek Mutalik and The BIOFAB Team at biofab.org.
NOTE: this version was later tested with riboswitch PDB Part:BBa_J100255 (containing promoter P5 and RBS BD18) which was too leaky to be useful. Therefore, it might be best to use promoter P9 and BD10 instead in subsequent applications of tClone Tet. See Part:BBa_J100279 and its experience page.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 958
Illegal BsaI.rc site found at 42