Overview

This year, our parts modeling focuses on predicting the result of the productivity of MSMEG_5998(BBa_K2382001) and Thioredoxin-MSMEG_5998 fusion protein(BBa_K2382009). Since our anti-aflatoxin protein could be used widely in many applications, such as antidote, and feed additive(see more in our human practice page), it is an indispensable important part to our project to have this data when we mass-produce our anti-aflatoxin protein in future work.

Material and Method

We transformed the plasmids that contained MSMEG_5998(BBa_K2382001) and Thioredoxin-MSMEG_5998 fusion protein(BBa_K2382009) respectively into competent cell E.coli BL21. After cultured overnight, measure the ABS600 and diluting the LB medium to O.D.=0.1. Then incubate at 37℃, 150 rpm until the O.D. of the samples reach 0.4 to 0.6 . Add 80ul 100mM IPTG( final concentration : 0.4mM ) to 125 ml flask and return to 37°C. From then on, after measure the O.D. values, transfer 1 ml from the induced sample and centrifuge at maximum speed for 60 seconds at RT and remove supernatant at 0, 1, 2, 3, 4, 5, 6, 7, 8 hours and 0, 0.5, 1.0, 1.5, 2.0 ,2.5 , 3.0, 3.5, 4 hours. Then we use Western Blot mehtod to amalyze the quantaty of MSMEG_5998 at each time spot.

Result

Fig. I The growth curve of BL21 induced by IPTG from 0 to 4 hours. The concentration of BL21 reached stationary phase at 4 hours.

Fig. II The growth curve of BL21 from 0 to 8 hr. The concentration of BL21 reached stationary phase at 4 hours and then declined slightly.

Fig. III Cell lysates from E. coli BL21 with Synthetic MSMEG5998 from 0 to 8 hours and 0 to 4 hours were analyzed by Western blot. The amount of Synthetic MSMEG5998 increased consistently with time.