Difference between revisions of "Part:BBa K3482023"

Revision as of 18:09, 27 October 2020

pLPT234 repressilator plasmid(Potvin-Trottier, 2016)

This plasmid encodes for three transcription factors (Tet, cI, lacI) and three reporter genes (mVenus, CFP, mKate2)

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.

pLPT234 is composed of three fluorescent reporter genes (mKate, CFP and mVenus) regulated by three different promoters (tetR, cI and lacI). This feedback loop circuit controlled the expression of three reporter genes leading to the oscillatory expression of three colors.

Schematic representation of plasmid pLPT234 [BBa_K3482023]. 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 three fluorescent proteins. Each reporter oscillates regularly.

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

The plasmid pLPT234 was tested with the three fluorescent reporter genes, in combination with the two sponge plasmids BBa_K3482024 and BBa_K3482026. The two figures show the oscillatory expression of our reporter genes for 12 hours, respectively with the sponge plasmids pLPT145 and pLPT41.

Oscillatory pattern obtained by transforming E.coli DHL708 with BBa_K3482023 (pLPT234) and Part:BBa_K3482024 (pLPT145), which are respectively the repressilator plasmid and the sponge plasmid.The bacteria were diluted to the same OD so the fluorescence would not depend on the quantity of bacteria. All bacteria were initially synchronized with aTC or IPTG to start oscillations at the same time. A plate reader was used to measure OD and fluorescence for each reporter gene. Data shows the mean and the standard deviation of 3 replicates.

Fluorescence over time plot of E. coli DHL708 containing the repressilator pLPT234 and the sponge plasmid pLPT41 synchronized overnight with 177 nM of aTC

We can see that the combination of the repressilator plasmid pLPT234 with the sponge plasmid pLPT145 gave clear and stable oscillations that correspond to our expectations and our model. The second combination, with the plasmid pLPT234 and pLPT41 gave less clear and stable oscillations, but we still observed the oscillations of the three reporter genes.

Sequence and Features