Difference between revisions of "Part:BBa K4634009"

 
Line 5: Line 5:
 
pBV220 plasmid with mCherry gene at its downstream.
 
pBV220 plasmid with mCherry gene at its downstream.
  
−
Evaluation of heat response ability:
+
===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 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]
 
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 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]
Line 12: Line 12:
 
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.
 
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.
 
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.
 +
 +
<center><html><img src='https://static.igem.wiki/teams/4634/wiki/parts-registry/bba-k4634007.png' width='600px'></html></center>
 +
<center><html>Fig 1. Evaluation of temperature-dependent promoter<br>
 +
①A: mCherry expression level change under different temperature and heating time.<br>
 +
②B: mCherry expression level change under different temperature, heating time =1h.
 +
</html></center>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
Line 17: Line 23:
  
 
<!-- -->
 
<!-- -->
−
<span class='h3bb'>Sequence and Features</span>
+
===Sequence and Features===
 +
<span class='h3bb'></span>
 
<partinfo>BBa_K4634009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4634009 SequenceAndFeatures</partinfo>
  
Line 25: Line 32:
 
<partinfo>BBa_K4634009 parameters</partinfo>
 
<partinfo>BBa_K4634009 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
===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.<br>
 +
[2] Abedi, M.H., et al., Ultrasound-controllable engineered bacteria for cancer immunotherapy. Nature Communications, 2022. 13(1): p. 1585.<br>
 +
[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

Revision as of 05:42, 4 October 2023


Temperature Control Induction Reporter System

pBV220 plasmid 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 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