Part:BBa_K539281

rbs+crtZ(beta-carotene hydroxylase) under heat sensitive cI regulater

This part contains crtZ, which works as the zeaxanthin biosynthesis pathway when add beta-carotene as a substrate. crtZ is regulated by cI and cI regulated promoter. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. cI binding results in repression of transcription.
The overview of the Carotenoid pathway

When we increase the temperature to 42℃, the CI proteins BBa_K098997, the repressor of cI regulated promoter BBa_R0051, are fully denatured. Then the cI regulated promoter is activated and CrtZ BBa_I742158 is produced, converting the orange beta-carotene to yellow Zeaxanthin.

This biobrick can coorperate with BBa_K539151 included crtEBIY. We also made crtEBIY under temperature controlled system. The circuit is shown below.
Below 37 degree

Under 37 degree

In order to prove the Carotenoid Pathway is work in our circuit, we made a new protocol.
Experiment methods:
1.The cells were cultured in LB over night.
2.Transferred 200ul LB to fresh 200ml LB and incubated at 37°C.
3.After 8 hrs, when OD ( optical density) reaches 0.1, then switch the temperature to 30°C, 37°C, 42°C respectively and incubate for 24hrs.
4.Centrifuged at 4°C, 6000rpm. The pellets were added with 1 ml ddH20 and vortexed.
5.Moved into 1.5ml eppendorf. Centrifuged for 20minutes at 4°C, 14000rpm.
6.Added with 500ul acetone, and vortexed for 1hr to extract the pigments.


Here shows the result of our circuit. The pellets collected from cultured cells in LB at the respective temperature. (30°C, 37°C and 42°C) The host cells, DH5 α, were cotransformed with BBa_K539151 on psb3T5 and BBa_K539281 on psb4A5.

The left two showing yellow color represent the the achievement after incubate at 30°C. The middle two showing orange color represent the the achievement after incubate at 37°C.The right two showing beige color represent the the achievement after incubate at 42°C. In our design, it suppose to express only CrtEBI and produce Lycopene and show red color at 30℃.When the temperature is at 37°C, it suppose to produce orange beta-Carotene. Produce yellow Zeaxanthin at 42°C. It color isn’t as our respect because of the interruption caused by the color of E.coli itself. And it’s proved later. We Added with acetone to extract the pigments. The result is as follow.



Here shows red color of 30°C cultured E.coli in acetone solution, and corresponds to the pigment, Lycopene.



Here shows orange color of 37°C cultured E.coli in acetone solution, and correspond to the pigment, beta-Carotene.



Here shows yellow color of 42°C cultured E.coli in acetone solution, and correspond to the pigment, Zeathanthin.



Photo of the three eppendorfs based on a white background.



Photo of the six eppendorves based on a black background.
We can see red Lycopene is produced at 30°C , orange beta-carotene is present at 37°C and yellow Zeaxanthine is produced at 42°C. The results of Carotenoid synthesis Pathway display different colors in different steps, that verify our concept of Pathway Commander perfectly.

Please refer to the Experience to surf more:BBa_K539281:Experience

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 NgoMIV site found at 1492
  
• 1000
  COMPATIBLE WITH RFC[1000]