Difference between revisions of "Part:BBa K2290000"

Revision as of 00:43, 30 October 2017

Fluoride Riboswitch regulated Chloramphenicol Acetyltranferase Operon

This is a composite part that allows for regulating the expression of a gene of interest with the B. ceres fluoride riboswitch. The functional parts of the switch are the following:

BBa_J23100 – a constitutively active promoter from the Anderson collection.

BBa_K2290008 – The B. cereus riboswitch with an engineered terminator that results in less read through/background. (This part is highly similar to part BBa_K911003 which is a part that includes both the Lys promoter and the riboswitch).

BBa_K2290004 – This is a sequence for a strong RBS that doesn't appear to be in the iGEM repository.

BBa_K2290005 – This is an E. coli optimized chloramphenicol resistance gene that we had synthesized.

BBa_B0010 – This is the T1 terminator from E. coli rrnB, reportedly a strong terminator

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 31
Illegal NheI site found at 54
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 182
Illegal XhoI site found at 887
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 14: / Line 14: @@
 BBa_B0010 – This is the T1 terminator from E. coli rrnB, reportedly a strong terminator
-In theory, this part can be used to screen any transcriptional riboswitch or regulate the expression of any gene with the fluoride riboswitch.
- is designed such that a user can easily clone in a new promoter and riboswitch or a gene of interest with Gibson cloning. To clone in a new promoter/riboswitch cut with HindIII. To clone in a new gene, cut with XhoI. Our part uses the E. coli constitutively active promoter J23100
 <!-- Add more about the biology of this part here
 ===Usage and Biology===
+This part is designed to allow the delta-crcB E. coli strain (strain JW0619-1 from the Yale Coli Genetics Stock Center) to grow on chloramphenicol in the presence of activating concentrations of fluoride (above 75uM). In theory, this part can be used to screen any transcriptional riboswitches by looking for growth on chloramphenicol in the presence of a ligand. To use CHOP in this manner, order an E.coli promotor riboswitch combination with appropriate Gibson overhangs, cut with HindIII to linearize the vector, and do Gibson. We also designed the vector so that genes besides the fluoride riboswitch can be swapped out easily. In this case cut with XhoI.
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 <span class='h3bb'>Sequence and Features</span>