Part:BBa_K2572001
Tcl42 thermal sensitive cl repressor
TcI42 is a high-performance thermal bioswitch. It is an upshifted variant of a well-known temperature-sensitive variant of the bacteriophage λ repressor cI (mutant cI857, here referred to as TcI) acting on a tandem pR–pL operator–promoter. The potential utility of these devices in three distinct in vivo scenarios relevant to mammalian microbial therapy is now well demonstrated. These scenarios include spatially selective activation within a mammalian host using focused ultrasound, sensing and response to a fever, and self-destruction at ambient temperatures to prevent environmental contamination after leaving the intended host. This coincides largely with our team’s work.
In our project we characterized its threshold temperature and sensitivity. Downstream gene expression is not detected at 37 degrees Celsius while it is bright at 39 degrees Celsius. This diagrees a little with the work of Piraner et al.(Piraner et al., 2016) which reports the threshold at 41-42 degrees Celsius.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Improvement
Group：UCAS-China
Characterizetion
In 2019, UCAS-China developed a collection of thermosensitive parts with high-performance, versatility and robustness. Based on TCI transcription factor family and TlpA family, we collected five TCI and TlpA mutants and used sfGFP as reporter to build some heat-inducible ON-switches, which can open gene expression under high temperature. To characterize them quantitatively, we first characterized the performance of them by flow cytometer in Top10 strain.
As shown in Figure 1, most of the transcription repressors show sharp thermal transitions, especially TCI and TCI42, with more than 100-fold induction within 10 degrees Celsius. Their impressive performances make them candidate parts for our further circuit design.
What’s more, we also tested these heat-inducible ON-switch in the chassis E.coli Nissle 1917, a probiotic with more than 100 years of medical application, their robustness give us more confidence in the stability and preciseness of our ark. The result is shown in Figure 2.
Reference
D. I. Piraner, A. Farhadi, H. C. Davis, D. Wu, D. Maresca, J. O. Szablowski, M. G. Shapiro, Going deeper: Biomolecular tools for acoustic and magnetic imaging and control of cellular function. Biochemistry 56, 5202–5209 (2017).
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