Difference between revisions of "Part:BBa J100419"
 
Line 12: Line 12:
  
 
rClone mScarlet (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 and GFP with the new RBS or riboswitch. Transcription will be initiated in the direction of the mScarlet coding sequence. The level of expression of mScarlet will depend on the efficiency of the newly cloned RBS or riboswitch.
 
rClone mScarlet (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 and GFP with the new RBS or riboswitch. Transcription will be initiated in the direction of the mScarlet coding sequence. The level of expression of mScarlet will depend on the efficiency of the newly cloned RBS or riboswitch.
 +
 +
When designing oligonucleotides for use with rClone Red, make sure they result in 5' overhang sticky ends that are CGAC (left) and GCGG (right).  Also make sure the oligonucleotides do not contain binding sites for BsaI. Finally, make sure the RBS element ends immediately before the GCGG right sticky end.  This will ensure a spacing of 6 bases between the RBS and the ATG start codon of RFP.  Below is an example.
 +
 +
<center>
 +
[[File:Oligos_for_rClone_Red.png|500px|]]
 +
</center>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 14:39, 5 August 2018


rClone mScarlet

This is the same sequence as the original rClone Red plasmid, but this plasmid has the mScarlet fluorescent protein inserted in place of the RFP. rClone Red: https://parts.igem.org/Part:BBa_J119384 mScarlet: https://parts.igem.org/Part:BBa_J100398

J100419 map.png

rClone mScarlet (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 and GFP with the new RBS or riboswitch. Transcription will be initiated in the direction of the mScarlet coding sequence. The level of expression of mScarlet will depend on the efficiency of the newly cloned RBS or riboswitch.

When designing oligonucleotides for use with rClone Red, make sure they result in 5' overhang sticky ends that are CGAC (left) and GCGG (right). Also make sure the oligonucleotides do not contain binding sites for BsaI. Finally, make sure the RBS element ends immediately before the GCGG right sticky end. This will ensure a spacing of 6 bases between the RBS and the ATG start codon of RFP. Below is an example.

Oligos for rClone Red.png

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 923
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 923
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 923
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 923
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 923
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 859
    Illegal BsaI.rc site found at 52