Part:BBa_K2290000
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 BBa_K911003, but the BBa_K911003 sequence 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
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 31
Illegal NheI site found at 54 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 182
Illegal XhoI site found at 887 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
2018 East Chapel Hill iGem:
We added new high quality experimental characterization to part BBa K2290000. Our procedure was as follows:
Day 1:
1. Transform ΔCRCB with plasmids by heat shock
2. Take 1 µl of plasmid and 20µL of cells
3. Place in 42 degree C water bath for 45 seconds
4. Cool on ice for 3 minutes
5. 'Shake’ at 37 degrees C for 1 hr
6.Make starter culture for o/w LB Amp
Day 2:
1. Grow e-coli on LB agar plates with 50 µ/mg concentration of chloramphenicol at varied fluoride concentrations.
2. Serial dilutions on agar plates
The image below demonstrates growth of ΔcRCB coli on plates of 50µ/mg chloramphenicol at concentrations of 0uM-100uM of fluoride. The left most column is part BBa K2290000. Our results showed more growth of ΔcRCB E. coli than what was previously documented by the 2017 East Chapel Hill iGem team. It is clear that growth by BBa K2290000 is dependent on the presence of fluoride.
//plasmidbackbone/libraryscreening
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