Difference between revisions of "Part:BBa K395704:Experience"

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<h1>Team WashU iGEM 2012:</h1>
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<h1>Team WashU iGEM 2012 Characterization of Part BBa_K39504:</h1>
 
The construct that Tokyo Tech built is composed of two parts: <br>
 
The construct that Tokyo Tech built is composed of two parts: <br>
 
1. CrtZ which is needed to cleave beta-carotene into zeaxanthin.  This component is repressed in the presence of glucose and <br> induced in the presence of arabanose through a pbad/arac promoter. <br>
 
1. CrtZ which is needed to cleave beta-carotene into zeaxanthin.  This component is repressed in the presence of glucose and <br> induced in the presence of arabanose through a pbad/arac promoter. <br>
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It can be observed from the graph that the groups with 2% arabanose are inhibited in their growth throughout the time measurements were taken, both with and without IPTG.  It can also be seen that the un-induced group had the highest OD600 at 3.75hrs suggesting that overproduction of the enzymes coded for by BBa_395704 does have a deleterious effect on the cells.  
 
It can be observed from the graph that the groups with 2% arabanose are inhibited in their growth throughout the time measurements were taken, both with and without IPTG.  It can also be seen that the un-induced group had the highest OD600 at 3.75hrs suggesting that overproduction of the enzymes coded for by BBa_395704 does have a deleterious effect on the cells.  
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While growth rate is important for the successful production of our target compounds, what is more important is the net amount of zeaxanthin made by the cells. To measure this we let the cells grow for 14hrs, towards the end of log phase, and measured cell denisty and amount of carotenoid produced.  The total carotenoids produced was measured by extracting pigments for a set volume of cells and measuring their absorbance at 450.  From this number we were able to calculate an approximate number of molecules of zeaxanthin produced per cell. 
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Latest revision as of 23:48, 1 October 2012

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K395704

Data sheet for zeaxanthin synthesis BioBricks (This part applies to ①)

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


registry-zea-1
figure1







Fig. 1.1.8.1 zeaxanthin pellet and plate
figure1
Fig. 2-1-1-3.TLC.jpg


















User Reviews

UNIQ7e044b8d9f3301af-partinfo-00000000-QINU UNIQ7e044b8d9f3301af-partinfo-00000001-QINU



Team WashU iGEM 2012 Characterization of Part BBa_K39504:

The construct that Tokyo Tech built is composed of two parts:
1. CrtZ which is needed to cleave beta-carotene into zeaxanthin. This component is repressed in the presence of glucose and
induced in the presence of arabanose through a pbad/arac promoter.
2. All the enzymes needed to produce beta-carotene from FPP in E. coli. Expression of these enzymes is regulated by a lacI repressed promoter, which is induced in the presence of lactose.
can be viewed below.





It can be observed from the graph that the groups with 2% arabanose are inhibited in their growth throughout the time measurements were taken, both with and without IPTG. It can also be seen that the un-induced group had the highest OD600 at 3.75hrs suggesting that overproduction of the enzymes coded for by BBa_395704 does have a deleterious effect on the cells. While growth rate is important for the successful production of our target compounds, what is more important is the net amount of zeaxanthin made by the cells. To measure this we let the cells grow for 14hrs, towards the end of log phase, and measured cell denisty and amount of carotenoid produced. The total carotenoids produced was measured by extracting pigments for a set volume of cells and measuring their absorbance at 450. From this number we were able to calculate an approximate number of molecules of zeaxanthin produced per cell.