Part:BBa_K3482025
pLPT119 repressilator plasmid(Potvin-Trottier, 2016)
This plasmid codes for a synthetic circuit composed of the transcription factors tetR, cI and lacI. It also codes for a constitutively expressed CFP reporter gene, and the mVenus reporter gene under the control of pTetO1
Usage and Biology
The repressilator is a synthetic regulatory network of at least three genes in a plasmid that repress each other in a feedback loop. These genes are cyclically expressed one after the other in a loop at regular intervals over time. This property of cyclic expression of each gene allows us to produce our anti-cancer drug regularly for cancer treatment. For our system to work efficiently, the sponge plasmid is applied together with the repressilator to smooth out the observed oscillations by buffering excess proteins produced by the main repressilator.
pLPT119 has the same composition as the repressilator system present on the plasmid pLPT234, with one fluorescent reporter gene less (mKate2 and its corresponding promoter were removed). This is then a two-reporter system, of which one is constitutively expressed and one oscillates.
(A) Schematic representation of plasmid pLPT119 [BBa_K3482025]. The promoters are represented by the arrows. The repressor genes are represented in grey. The reporter genes are colored according to the absorbance wavelength of their proteins. In this system, tetR is inhibiting cI, which is inhibiting lacI, which itself inhibits tetR back. (B) Model of the oscillation of our two fluorescent proteins. CFP is expressed in a constant manner, mVenus is oscillating as before.
All of the plasmids presented here were obtained and ordered from Addgene. They were all made available by the Professor Johan Paulson. All the plasmids come form this article Potvin-Trottier, L., Lord, N., Vinnicombe, G. et al. Synchronous long-term oscillations in a synthetic gene circuit. Nature 538, 514–517 (2016). https://doi.org/10.1038/nature19841.
Characterization
We transformed the pLPT119 repressilator plasmid with the pLPT41 sponge plasmid into E. coli Nissle to check whether we obtained oscillations. The two figures show the oscillatory expression of our reporter genes for 12 hours, with the sponge plasmid pLPT41 in both E.coli Nissle 1917 and DHL708, respectively.
Fluorescence over time plot of E. coli Nissle 1917Δclb containing plasmids pLPT119 and pLPT41. The double lines for each reporter gene represent two biological replicates.
Fluorescence over time plot of E. coli DHL708 containing plasmids pLPT119 and pLPT41.
In the pLPT119 repressilator we only expected the mVenus (green) reporter gene to have an oscillatory expression pattern. CFP (blue) was under the regulatory control of a constitutive promoter which meant we expected the expression of the reporter gene to be constant and follow the change in OD. These results suggest that the pLPT119 repressilator and pLPT41 sponge plasmid work in both E. coli DHL708 and E. coli Nissle 1917Δclb
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 75
Illegal EcoRI site found at 831
Illegal EcoRI site found at 1000
Illegal EcoRI site found at 1096
Illegal EcoRI site found at 6309
Illegal EcoRI site found at 7647
Illegal EcoRI site found at 8508
Illegal XbaI site found at 1855
Illegal XbaI site found at 5546
Illegal XbaI site found at 6511
Illegal XbaI site found at 7842
Illegal SpeI site found at 3870
Illegal PstI site found at 837
Illegal PstI site found at 6395
Illegal PstI site found at 7726 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 75
Illegal EcoRI site found at 831
Illegal EcoRI site found at 1000
Illegal EcoRI site found at 1096
Illegal EcoRI site found at 6309
Illegal EcoRI site found at 7647
Illegal EcoRI site found at 8508
Illegal SpeI site found at 3870
Illegal PstI site found at 837
Illegal PstI site found at 6395
Illegal PstI site found at 7726 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 75
Illegal EcoRI site found at 831
Illegal EcoRI site found at 1000
Illegal EcoRI site found at 1096
Illegal EcoRI site found at 6309
Illegal EcoRI site found at 7647
Illegal EcoRI site found at 8508
Illegal BamHI site found at 849
Illegal BamHI site found at 1117
Illegal BamHI site found at 5540
Illegal XhoI site found at 8588
Illegal XhoI site found at 8669
Illegal XhoI site found at 8697 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 75
Illegal EcoRI site found at 831
Illegal EcoRI site found at 1000
Illegal EcoRI site found at 1096
Illegal EcoRI site found at 6309
Illegal EcoRI site found at 7647
Illegal EcoRI site found at 8508
Illegal XbaI site found at 1855
Illegal XbaI site found at 5546
Illegal XbaI site found at 6511
Illegal XbaI site found at 7842
Illegal SpeI site found at 3870
Illegal PstI site found at 837
Illegal PstI site found at 6395
Illegal PstI site found at 7726 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 75
Illegal EcoRI site found at 831
Illegal EcoRI site found at 1000
Illegal EcoRI site found at 1096
Illegal EcoRI site found at 6309
Illegal EcoRI site found at 7647
Illegal EcoRI site found at 8508
Illegal XbaI site found at 1855
Illegal XbaI site found at 5546
Illegal XbaI site found at 6511
Illegal XbaI site found at 7842
Illegal SpeI site found at 3870
Illegal PstI site found at 837
Illegal PstI site found at 6395
Illegal PstI site found at 7726
Illegal NgoMIV site found at 5322 - 1000COMPATIBLE WITH RFC[1000]
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