Part:BBa_K3086009
SCRIBE (RpoB) System
This is our validated part. SCRIBE( Synthetic Cellular Recorders Integrating Biological Events) is a tool that utilizes modified retrons that have been transformed into bacterial cells to produce single-stranded DNA in response to a certain stimulus. The ssDNA is incorporated into the genome using the replication system of the bacteria. This technique mutates the bacteria to have rifampicin resistance.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1877
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1877
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1282
Illegal XhoI site found at 516 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1877
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1877
- 1000COMPATIBLE WITH RFC[1000]
Verifying the SCRIBE system
For the first part of our project, we are testing to see if Fahim Farzadfard and Timothy K. Lu’s paper, “Genomically encoded analog memory with precise in vivo DNA writing in living cell populations” rings true in regards to whether the SCRIBE system works or not. In order to check for the SCRIBE system, we incorporated a target sequence that gave rise to specific antibiotic resistance. rpoB gives rise to rifampicin resistance and rpsL gives rise to streptomycin resistance. If the SCRIBE system is working correctly, then the target sequence was successfully incorporated into the chromosomal DNA of the E.coli cell. Therefore the host cell will express the corresponding antibiotic resistance.
Rifampicin serial dilution plates
Experiments 1-4
We checked to see if the rpoB target sequence was incorporated into the pFF745 plasmid and whether it gave the host cells rifampicin resistance. This was proved by the growth of colonies on the rifampicin plates.
Experiment 1
The first experiment with E.coli represents the negative control therefore proving that pFF745 does not normally express rifampicin resistance.
Experiments 2, 3, and 4
Experiments 2, 3 and 4 are all E. coli with the rpoB target sequence. Since they were able to grow on the rifampicin plate, we can conclude the SCRIBE system successfully worked.
The sequencing data shows that the rpoB target was successfully put into the plasmid. The sequencing reaction is on the bottom and the designed plasmid sequence is on the top. The gray bars at the top show that the whole sequence matches.
Goal: We wanted to verify the host cells’ resistant to rifampicin antibiotic came solely from the rpoB mutation and not from another source.
rpoB is specific to rifampicin resistance while rspL is specific to streptomycin resistance. In order to rule out the possibility that other cells without the rpoB sequence can grow on rifampicin plates, we grew rpsL cells (with C and D orientation) on those plates. We patched the C and D cells on dilution plates, comparing cells that were and were not induced with IPTG (IPTG induces the SCRIBE system). As shown on the plate below, E. coli cells with rpsL as the target sequence were not able to grow on rifampicin plates. This supports our claim that only rpoB cells grew on the rifampicin plates.
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