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Tcl857 repressor with PL and PR promoter.

Evaluation of heat response ability:

PL and PR promoter are typical elements regulated by temperature and ubiquitously employed in bacterial expression systems. The repressor, Tcl857, is a temperature sensitive mutant of phage λ. Under normal condition, Tcl857 act as dimers that bind to the operator site, hindering the occurrence of transcription. When the temperature rises to about 42 °C, the repressor is thus denatured and the transcription happens.[1] To visualize the thermal dependence of this composite part, mCherry gene is connected at its downstream.

Fig 1A demonstrates the mCherry expression levels under the two variables, heat shock temperatures and times. Compared to 37 °C, the increase of heat shock temperature significantly activated the promoter activity. Among the conditions tested, we detect strong signal under 41-43 °C. Rising the temperature to 45 °C, however, decreased mCherry containment. This may be resulted from the extreme high temperature which caused physiological damage to the bacteria cells. Then, we set up a more delicate temperature slope from 41 °C to 43 °C, setting the heating time for 1hour, and the results are elicited in Fig 1B. We found that the fold change in 42 and 43 °C were similarly significant, while 41 °C is much lower, which is consist with the data of previously published research[2, 3], which reported that Clt857 responded to a 42 Celsuis heat shock.

Fig 1. Evaluation of temperature-dependent promoter
①A: mCherry expression level change under different temperature and heating time.
②B: mCherry expression level change under different temperature, heating time =1h.

Sequence and Features

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References

[1] Ju, L.W., et al., GeneDn: for high-level expression design of heterologous genes in a prokaryotic system. Bioinformatics (Oxford, England), 1998. 14(10): p. 884-885.
[2] Abedi, M.H., et al., Ultrasound-controllable engineered bacteria for cancer immunotherapy. Nature Communications, 2022. 13(1): p. 1585.
[3] Chen, Y., et al., Spatiotemporal control of engineered bacteria to express interferon-γ by focused ultrasound for tumor immunotherapy. Nature Communications, 2022. 13(1): p. 4468