Translational_Unit

Part:BBa_K395705:Experience

Designed by: Yumiko Kinoshita   Group: iGEM10_Tokyo_Tech   (2010-10-06)
Revision as of 23:18, 6 November 2010 by ODDY (Talk | contribs) (→‎Applications of BBa_K395705)

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Applications of BBa_K395705

These parts contain crtW (β-carotene ketolase) under inducible promoter control. This enzyme is responsible for the conversion of zeaxanthin into astaxanthin. Our team designed two BioBricks related to this conversion. Characterization of BBa_K395706 has been performed in E. coli strain MG1655. (→[http://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter more information])


registry-asta-1

registry-asta-2

Fig. 2-1-11 astaxanthin pellet and plate
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Fig. 2-1-13 allTLC







Material

E. coli  : strain MG1655

reagent  : acetone, methanol, chloroform, hexane

TLC plate  : silicagel 60F254 culture

1. The cells were cultured over night.

2. 50ul bacterial solution was taken from culture prepared in 1 and moved into 50ml LB culture with appropriate antibiotic. Inducer was added, if necessary.

3. The cells were incubated at 37°C, 24hour. gathering E. coli and extract pigment

4. After the O.D reach 2.0, culture was centrifuged for 10minutes at 4°C, 6000rpm,10min

5. Supernatant was removed by decanting. The remainder was added with 1 ml ddH20 and vortexed.

6. Moved into 2ml tube by using P1000 pipetter at least twice.

7. Solution is centrifuged for 20minutes at 4°C, 14000rpm

8. Supernatant was removed completely using pipetter. About 100ul of pellet is obtained.

9. Pellet is added with 500ul acetone, and vortexed for 10 minutes to extract the pigment.

10. Solution is centrifuged for 20minutes at 4°C, 14000rpm

11. Acetone solution was replaced into 2 ml tube. vacuum and concentrate

12. Acetone was removed using rotary evaporator.

13. 50 ul of Chloroform/Methanol(weight ratio of 9:1) solution was added.

14. Sample was vortexed and spinned down.

15. The water layer was removed. TLC

16. Spot the sample onto TLC silica gel plate, and developed by acetone/hexane(weight ratio of 1:3) solution. 17. Spot was observed under visible light. Retrieved from "http://2010.igem.org/Team:Tokyo_Tech/Project/Apple_Reporter" Powered by MediaWiki Attribution-Share Alike 3.0 Unported

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