Overview
This year, we have successfully:
- replicated Masp2_2, Masp2_1, Masp_1_102, Masp1_1 and NT-2Rep-CT-SpyTag.
- made two recombinant plasmids, Masp1_102 with pET-SUMO and NT-2Rep-CT-SpyTag with pET-22b(+)
- expressed proteins from NT-2Rep-CT-SpyTag
- purified and concentrated the spidroins
- synthesised spider silk from the spidroins expressed
Replication
Five DNA including Masp2_2, Masp2_1, Masp1_102, Masp1_1 and NT-2Rep-CT-SpyTag and three plasmids, including pET-SUMP, pET-HisB and pET-22b(+) are successfully replicated.
Cloning
To make the recombinant plasmids, we carried out digestion and ligation. And the recombinant plasmids were transformed in E. coli Top10. Colony PCR were carried out for those five plasmids. All plasmids have at least one colony that shows the correct band. Liquid culture and miniprep were carried out for those colonies with positive results in colony PCR. All show correct bands in gel electrophoresis. We then sent all of the samples to the gene company to do sequencing. Only Masp1_102 and NT-2Rep-CT-SpyTag have ideal sequencing results. For those two recombinant plasmids, transformation has been done one more time, but the E. coli should be BL21 for better expression.
XbaI and HindIII digestion check for Masp1_102(~1.2kb)
Here shows eight digestion products of Masp1_102 samples
Four of them (lines 2,4,6 and 7) shows positive results
XbaI and HindIII digestion check for Masp1_1(~1.2kb)
lane 1 is digested and lane 2 does not
T7 primers for screening PCR
with size of ~1.2kb
Expression
After transformation, we selected the colonies to carry out protein expression with inducer IPTG. After that, we did SDS-PAGE for both samples. Both results are not good.
As the protein samples used for SDS-PAGE is only from 2ml of LB culture solution, the spridrion concentration is too low and undetectable. And then, the 35kDa band in the NT-2 Rep-CT-SpyTag sample may be caused by non-target protein. In order to figure out the problem, the NT-2Rep-CT-SpyTag sample proceeded to protein purification. The 2L LB culture solution was then purificated. Around 60ml of protein solution is finally produced and another SDS-PAGE has been done.
For the NT-2Rep-CT-SpyTag, we proceed to protein purification. And run SDS-PAGE. The results are satisfactory.
During purification we collected the samples at different steps for SDS-PAGE. Compared to the other line, the last two lines obviously show less proteins and bands with 35kDa are formed again. This indicates that His-tag gene is work and the 35kDa band should represent the presence of spridroin.
Silk Fabrication
The spider silk was successfully formed from the spridroin by pushing the syringer.