Difference between revisions of "Part:BBa J119389"

Latest revision as of 19:29, 6 April 2018

rClone Blue: Device for GGA Cloning and Testing RBS elements and Riboswitches

rClone Blue (see Eckdahl et al. 2017) allows users to clone and test new RBS elements and riboswitches without gel purification or other preparation of DNA. It is a destination vector for Golden Gate Assembly (GGA) using BsaI and ligase. A new RBS 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 RBS or riboswitch. Transcription will be initiated in the direction of an Amil CP blue chromoprotein coding sequence. The level of expression of blue chromoprotein will depend on the efficiency of the newly cloned RBS or riboswitch.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1404
Illegal AgeI site found at 1512
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 849
Illegal BsaI.rc site found at 42

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 <partinfo>BBa_J119389 short</partinfo>
-rClone Blue (see [http://www.jyi.org/issue/rclone-a-synthetic-biology-tool-that-enables-the-research-of-bacterial-translation/ Eckdahl et al. 2017]) allows users to clone and test new RBS elements and riboswitches without gel purification or other preparation of DNA. It is a destination vector for Golden Gate Assembly (GGA) using BsaI and ligase. A new RBS 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 RBS or riboswitch. Transcription will be initiated in the direction of the RFP coding sequence. The level of expression of Blue Chromoprotein will depend on the efficiency of the newly cloned RBS or riboswitch.
+rClone Blue (see [https://www.jyi.org/2017-march/2017/5/1/rclone-a-synthetic-biology-tool-that-enables-the-research-of-bacterial-translation/ Eckdahl et al. 2017]) allows users to clone and test new RBS elements and riboswitches without gel purification or other preparation of DNA. It is a destination vector for Golden Gate Assembly (GGA) using BsaI and ligase. A new RBS 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 RBS or riboswitch. Transcription will be initiated in the direction of an Amil CP blue chromoprotein coding sequence. The level of expression of blue chromoprotein will depend on the efficiency of the newly cloned RBS or riboswitch.
 <center>
 [[File:RClone_Blue.png]]