Team:CSMU NCHU Taiwan/Model/Parts
Parts model
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.
Discussion
1. According to the data shown above, the growth curve of E.coli BL21 with Synthetic MSMEG_5998 reached the ceiling when the O.D. value was approximately at 2 while the amount of Synthetic MSMEG_5998 were still increasing.
2. Though the amount of Synthetic MSMEG_5998 increased consistently with time, we could not jump to conclusions that it was proper to incubate E.coli as long as possible. Another consideration was the time it would take. Just as our expected, it growed fast at the first 2.5 hours. That’s why we also chose 2.5hr after induced by IPTG when we extracted Synthetic MSMEG_5998 from total cell lysate in other experiments.
3. Based on previous experience, if the E.coli was incubated over 4 hours, the protein that it expressed may be degraded or mis-folded, leading to malfunction. As a result, it was also an important issue for this modeling. However, because of the lack of F420, we did not have the chance to check the enzyme activity of each time spot. It was still unknown whether the titer of the Synthetic MSMEG_5998 would change or not and awaited further research.
