Difference between revisions of "Part:BBa K4634009"

Latest revision as of 17:12, 6 October 2023

Temperature Control Induction Reporter System

PL and PR promoter with mCherry gene at its downstream.

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 Celsius, the repressor is thus denatured and the transcription happens.[1] Here mCherry gene is connected at its downstream for visualization of thermal dependence of the plasmid.

Fig 1A demonstrates the mCherry expression levels under the two variables, heat shock temperatures and times. Compared to 37 Celsius, the increase of heat shock temperature significantly activated the promoter activity. Among the conditions tested, we detect strong signal under 41-43 Celsius. Rising the temperature to 45 Celsius, 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 Celsius to 43 Celsius, setting the heating time for 1hour, and the results are elicited in Fig 1B. We found that the fold change in 42 and 43 Celsius were similarly significant, while 41 Celsius 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

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 1324
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 1324
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 838
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 1324
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 1324
1000
COMPATIBLE WITH RFC[1000]

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

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4634009 short</partinfo>
-pBV220 plasmid with mCherry gene at its downstream.
+PL and PR promoter with mCherry gene at its downstream.
 ===Evaluation of heat response ability:===
-pBV220, which contains 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 &#955;. 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 Celsius, the repressor is thus denatured and the transcription happens.[1]
+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 Celsius, the repressor is thus denatured and the transcription happens.[1]
 Here mCherry gene is connected at its downstream for visualization of thermal dependence of the plasmid.