Difference between revisions of "Part:BBa K539281"

Revision as of 18:13, 5 October 2011

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 https://parts.igem.org/Image:Crt-2.jpg
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 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