Part:BBa_K539151
promoter+crtEBI+RNA thermometer(FourU)+crtY+terminator
This part is a generator of crtEBIY. Promoter BBa_J23103[1] + CrtEBI(BBa_K274100[2]) 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 [3]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[4] is activated . CrtY BBa_K118008[5] is translated, which convert red Lycopene to orange beta-Carotene.
The circuit BBa_K539281[6] 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.
Here shows the result of our circuit. The colored pellets represent the achievement after incubate in 30°C(left two), 37°C(middle two), 42°C(right two) for 24 hours respectively.
In order to decrease the interruption caused by the color of E.coli itself, we should extract the pigment with organic solvent since the three pigments are hydrophobic. So Acetone is added and vortexed to form a homogeneous colored solution.
Here shows color of 30°C cultured E.coli , and corresponds to the pigment, Lycopene.
Here shows color of 37°C cultured E.coli, and correspond to the pigment, beta-Carotene.
Here shows color of 42°C cultured E.coli, and correspond to the pigment, Zeaxanthin.
Photo of the three eppendorfs based on a white background.
Photo of the three eppendorves based on a black background.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2017
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
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