Team:UESTC-China/Notebook
June
Based on the available synthetic plasmids, we arranged the work of each member and carried out preliminary preparations such as purchasing necessary equipment and drugs. In addition, according to the pichia pastoris expression book and the actual laboratory
situation, we have designed a complete set of experimental flow schemes and set a weekly schedule to push our experiments forward.
July
7.1-7.7
We prepared competent escherichia coli and transfered our six target plasmids EG1, EGL7, XynB, LipLac, Scaford, SdbA synthesized by the company into E.coil. Then we use low-salt(LB) medium to culture our E. coli in order to get enough bacterial deposits
to extract our plasmids.
At the same time, we consulted a great number of literatures concerning modified enzymic preparations and bacterial preparations to determine which preparation we should use for subsequent deinking validation.
7.8-7.14
We extracted target plasmids from our escherichia coli bacterial deposits and we obtained a large number of target plasmids. We used agarose gel electrophoresis to verify our target plasmids and we used ethanol to precipitate the successful plasmids.
At the same time, we began to explore the conditions for the cultivation of Pichia pastoris and then cultivated Pichia pastoris. We finally obtained a batch of pure Pichia pastoris which was found to be free from bacterial contamination by microscopic
examination.
7.15-7.21
We began to explore the electrotransformation conditions, and tried to transfer our EG1, EGL7, XynB, LipLac, Scaford, SdbA plasmids into a new batch of Pichia pastoris and verified their successful expression of related proteins.
At the same time, we began to explore methods to detect the deinking activity of commercial enzymes.
7.22-7.31
We were stuck in our experiment for the reason that every time we cultured pichia pastoris after electrotransformation, miscellaneous bacteria always appeared. We finally decided to eliminate our lab completely by using potassium permanganate and formaldehyde.
During formaldehyde elimination in the lab, we started writing the framework of our wiki and drew up subsequent experimental arrangements. In addition, we also used the laboratory downstairs to conduct normal deinking verification experiments and successfully
verified that cellulase and xylanase have a decent deinking effect.
August
8.1-8.7
We were able to re-enter our old lab after testing formaldehyde levels to meet national standards and we used our original plasmids and restarted to culture a new batch of pichia pastoris in order to restart the electrotransformation. We finally succeeded
in avoiding contamination by miscellaneous bacteria and we successfully verified the target bands by colony PCR.
At the same time, we began to verify the deinking effect of laccase and lipase on paper and explore the synergistic effect between enzymes. However, we found that the deinking effect of laccase and lipase was not satisfactory.
8.15-8.21
We consulted our teacher and learned that the transformant would produce a large number of false positives and our teacher suggested that we could use three-rounds 96-well plates for screening. We adopted our teacher's suggestion and screened a batch
of new pichia pastoris through electrotransformation in 96-well plate.
At the same time, we have improved our deinking verification method and began to process a new round of deinking data. We used the line chart and histogram to show our processed data and successfully verified the correlation between the enzymes and deinking
effects.
8.22-8.28
We took our positive transformants screened by 96-well plates and fermented them. As before, we collected the bacterial solution from 1 to 4 days of fermentation. But this time we first used dot blot to verify that our protein was successfully expressed
and then detected the enzyme activity of the target enzymes. This time we successfully verified the enzyme activity of our cellulase and lipase.
8.29-8.31
We began continuous fermentation and tried to detect laccase and xylanase activity at the same time we designed a new experimental method to verify the deinking effect of laccase and lipase. At the same time, we successfully produced the second generation
of deinking machine.
September
9.1-9.7
For those former pichia pastoris that dot blot tested successfully, we used SDS-PAGE and Western Blot to further verify our protein expression results, but it didn't work out so well. Our analysis is that the target protein of the previously preserved
fermentation liquor has been degraded. Therefore, we started to re-ferment a new batch of pichia pastoris.
9.8-9.14
After a lot of trial and error, we finally verified the target protein bands with western Blot and we finally determined the optimal deinking time of the core enzymes cellulase and xylanse and successfully produced the third generation of deinking machine.
9.15-9.21
The work of deinking verification has basically been completed and we set out to verify the binding of scaffold proteins to enzymes but until now we have not detected the xylanase and laccase enzyme activity.
9.22-9.31
We carried out some supplementary experiments for our deinking work. We tried to use fermented supernatant for deinking verification, but the result was not good. At the same time, we carried out a lot of fermentation to get enough target protein.
October
10.1-10.5
We finally measured the xylanase activity by ammonium sulfate precipitation method of protein concentration and we began to verify the promoting effect of scaffolding on targeted protein.
10.6-10.8
We ultrasonically verified the expression and the activity of all our key enzymes and successfully tested their function.
10.9-10.21
We did some supplementary experiments to verify our design and theory and we began to write wiki and prepare for the final presentation.