Difference between revisions of "Part:BBa K4055001"
(→‎Usage and Biology)
 
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This is a similar design to [[Part:BBa_K3628024]] but split the T7RNAP between aa179 and aa180.
 
This is a similar design to [[Part:BBa_K3628024]] but split the T7RNAP between aa179 and aa180.
−
The split T7RNAP has little activity in a dark environment. When the two segments were exposed to blue light, they will bind together due to the photodimerization of VVD domain and exhibit full RNA polymerase activity.
+
The split T7RNAP has little activity in dark environment. When the two segments were exposed to blue light, they will bind together due to the photodimerization of VVD domain and exhibit full RNA polymerase activity.
 
So it can be used for constructing a photo sensor system with T7 promoter.  
 
So it can be used for constructing a photo sensor system with T7 promoter.  
  
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===Usage and Biology===
 
===Usage and Biology===
 
We inserted this part into a pSB4C5 backbone, and transformed it into the Escherichia coli DH5α host cell which had harbored a pSB1A3 plasmid containing a [[Part:BBa_K4055532|pT7-amilCP transcriptional unit]]. Then test it in tubes.
 
We inserted this part into a pSB4C5 backbone, and transformed it into the Escherichia coli DH5α host cell which had harbored a pSB1A3 plasmid containing a [[Part:BBa_K4055532|pT7-amilCP transcriptional unit]]. Then test it in tubes.
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+
[[File:T--Yucai_SZ--splitT7RNAP.png|center|Testing system for photosensitive T7RNAP]]
 
*Inoculate a colony into LB medium and culture overnight in dark (200 rpm, 37 ℃).
 
*Inoculate a colony into LB medium and culture overnight in dark (200 rpm, 37 ℃).
 
*Overnight cultures are inoculated 1:100 into two tubes with fresh LB medium. When cultured in shaking table, one tube is exposed to 480 nm blue light, and the other one is covered by tin foil, to protect it from light
 
*Overnight cultures are inoculated 1:100 into two tubes with fresh LB medium. When cultured in shaking table, one tube is exposed to 480 nm blue light, and the other one is covered by tin foil, to protect it from light
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*Centrifugate the sample tubes at 10 000 g for 30 seconds and observe the colors of bacterial precipitations
 
*Centrifugate the sample tubes at 10 000 g for 30 seconds and observe the colors of bacterial precipitations
  
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<b>Result</b>
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[[File:T--Yucai_SZ--K4055001andK4055532.jpeg|300px|thumb|center|The left was cultured under blue light, and the right was in dark]]
  
  

Latest revision as of 00:05, 22 October 2021


J23106-RBS-T7 RNA polymerase N 1~179-Vvd-RBS-Vvd-T7 RNA polymerase C 180~883

This is a similar design to Part:BBa_K3628024 but split the T7RNAP between aa179 and aa180. The split T7RNAP has little activity in dark environment. When the two segments were exposed to blue light, they will bind together due to the photodimerization of VVD domain and exhibit full RNA polymerase activity. So it can be used for constructing a photo sensor system with T7 promoter.


Usage and Biology

We inserted this part into a pSB4C5 backbone, and transformed it into the Escherichia coli DH5α host cell which had harbored a pSB1A3 plasmid containing a pT7-amilCP transcriptional unit. Then test it in tubes.

Testing system for photosensitive T7RNAP
  • Inoculate a colony into LB medium and culture overnight in dark (200 rpm, 37 ℃).
  • Overnight cultures are inoculated 1:100 into two tubes with fresh LB medium. When cultured in shaking table, one tube is exposed to 480 nm blue light, and the other one is covered by tin foil, to protect it from light
  • After 12 hours of shaking culture, 500 μL culture were sampled and transferred into 1.5 mL EP tube.
  • Centrifugate the sample tubes at 10 000 g for 30 seconds and observe the colors of bacterial precipitations

Result

The left was cultured under blue light, and the right was in dark


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 798
    Illegal AgeI site found at 1026
    Illegal AgeI site found at 1343
    Illegal AgeI site found at 1571
  • 1000
    COMPATIBLE WITH RFC[1000]