Part:BBa_K539151

promoter+crtEBI+RNA thermometer(FourU)+crtY+terminator

This part is a generator of crtEBIY. Promoter BBa_J23103 + CrtEBI(BBa_K274100) is submitted by team Peking 2010. Enzyme cassette CrtEBI (with individual rbs) converts colourless farnesyl pyrophosphate to red lycopene (via intermediates geranylgeranyl pyroiphosphate and phytoene). And CrtY converting lycopene to orange beta-carotene. Our team assemble BBa_K346090(promoter+crtEBI) and RNA thermometer+crtY+terminator. This RNA thermometer initiate translation at 37°C. The existence of RNA thermometer makes crtY translated at specific temperature. We construct another circuit BBa_K539281 included crtZ that can cooperate with circuit BBa_K539151.

If E.coli is incubating below 37℃, mRNAs cannot be translated by ribosomes, because temperature-sensitive RBS(ribosome biding site) BBa_K115002 forms the hairpin. A ribosome cannot bind to the RBS so that it cannot translate CrtY BBa_K118008. As a result, the E.coli only can produce CrtE, CrtB and CrtI BBa_K346090 which convert colorless Farnesyl pyrophosphate to red Lycopene.

When the temperature is at 37℃ or higher, the hairpin is denatured so temperature-sensitive RBS BBa_K115002 is activated . CrtY BBa_K118008 is translated, which convert red Lycopene to orange beta-Carotene.

The circuit BBa_K539281 made by our team includes the gene crtZ that is under 42℃ induced device.

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.


Figure 1. 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 andBBa_K539281 on psb4A5.
The left two showing yellow color represent the achievement after incubated at 30°C. The middle two showing orange color represent the achievement after incubated at 37°C.The right two showing beige color represent the achievement after incubated at 42°C.

In our design, it supposes to express only CrtEBI and produce Lycopene and show red color at 30℃.When the temperature is at 37°C, it supposes to produce orange beta-Carotene and produce yellow Zeaxanthin at 42°C. The color isn’t achieved our respect because of the interruption caused by the color of E.coli itself. And it’s proven later.
We added acetone to extract the pigments. The results are as following.



Figure 2. Here shows red color of 30°C modified E.coli in acetone solution , and corresponds to the pigment, Lycopene.



Fifure3. Here shows orange color of 37°C modified E.coli in acetone solution, and correspond to the pigment, beta-Carotene.



Figure 4. Here shows yellow color of 42°C modified E.coli in acetone solution, and correspond to the pigment, Zeaxanthin.



Figure 5.Photo of the three eppendorfs based on a white background.
The left showing red color represent the the achievement after incubate at 30°C. The middle showing orange color represent the the achievement after incubate at 37°C.The right showing yellow color represent the the achievement after incubate at 42°C.


Figure 6.Photo of the six eppendorfs based on a black background.


Comment 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.

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 2017
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1553
  Illegal NgoMIV site found at 1683
  Illegal NgoMIV site found at 4654
  Illegal AgeI site found at 768
  
• 1000
  COMPATIBLE WITH RFC[1000]