Characterization

Z-Construct

Saffron in a Kan is focused on the modification of carotenoids to produce compounds characteristic of saffron. The first step to doing this is optimizing the production of the carotenoid precursor, zeaxanthin, in our target species. Thankfully, Tokyo Tech
developed a biobrick which contains all the enzymes necessary to produce zeaxanthin in E. coli, BBa_395704. This is an enormous help in advancing our project, however the biobrick lacked the characterization needed for optimal production of zeaxanthin through utilization of its inducible promoters.

To this aim, we further charcterized this part for both our project and for furture teams working on a developing biobricks that depend on the synthesis of zeaxanthin in vivo.

Optimization of induction conditions

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. (protocol found here: link) From this number we were able to calculate an approximate number of molecules of zeaxanthin produced per cell.