Contribution for SHSBNU_China

iGEM 2019 team GreatBay_SZ designed a project to produce recombinant spidroins (silk proteins) from E.coli and spin them into silk. Our team SHSBNU_China this year concentrated on using microbial as a biological chasis to yield polymers, which has some similarity with the previous GreatBay_SZ project. According to this, we synthesized the sequence uploaded and constructed it onto pET28a(+) plasmid to repeat the experiments at first.

The new plasmid was named as pET28a(+)-NT-2Rep-CT, which was transfected into E.coli BL21(DE3) for efficient protein expression. However, we noticed two essential problems for GreatBay_SZ in their experiment:
1) They elucidated to suggest a complex growth medium that integrate a specific trace metal solution to cultivate the bacteria.
2) They found the spidroin continuely altered from soluble to insoluble state.
Our team SHSBNU_China discussed about the two problems, we thought that although the bacteria can grow in the given condition, but it could bring great inconvenience for the subsequent experiment. Especially when the silk is indeed used in the textile industry, is it safe for human beings and our environment? Besides, based on our experience in titin program this year, we believe the insoluble state can be improved by changing the expression condition.

According to the above discussion, we tried to cultivate the bacteria in standard LB culture, and we set the following conditions:
1) induction for 20 hours under 16℃
2) induction for 4 hours under 37℃

As we can see in the SDS-PAGE, spidroin can be expressed using standard LB culture, and although it precipitated in the inclusion body under 37℃, it became soluble after induction for 20 hours under 16℃. Based on these results, we believe we offered a new solution for the production of spidroin, which solves the problem of specific culture conditions and solubility.