Team:SIAT-SCIE/Results
Vector Construction
-Overview:
We are using pMD-19 plasmid backbone for CAHS and p15A plasmid backbone of DSUP.
At the beginning, we tried to use pET28a for protein extraction, but due to the unknown region on the sequencing, we gave it up.
Here, we will show the construction of pMD-19-pTet-tetR-CAHS plasmids, since p15A-pTac-sfGFP-Dsup was constructed by Bluepha.
The assembly method we choose was similar to Gibson assembly, it requires overlaps on both ends, and use exonuclease to cut out the ends for ligation.
-PCR:
We used PCR to add the overlapping fragments on our DNA fragment.
The result of it is shown by gel electrophoresis.
-Ligation
After gel-extraction, we added 0.06 pmol of DNA fragments and 0.03 pmol of backbone, incubated for 30 minutes under 37°C in PCR machine, the product was cooled in ice for three minutes, and immediately transform it into the DH5α Chemical Component cell.
-Clone PCR
After leaving the DH5α containing previous products in 37°C overnight, we carried out the Clone PCR to verify the ligation
The results are shown by gel electrophoresis.
According to this, we found that Dsup was not successfully inserted into the backbone. After several times, we decided to send it to Bluepha, for the construction of Dsup.
-Sequencing
Samples were sent to BGI for sequencing, the results are complementary to our design.
Protein Expression
-Inducer Adding
SDS Page
Unfortunately, the SDS-PAGE experiments were done badly, there were always mistakes, including sample contamination, failure in protein expression, faulty in stain and decolor, so we did not get a very reliable result on this.
BUT WE MADE IT!
DURING OUR LAST TRIAL ON OCTOBER 27TH
Desiccation
Dilution
In order to find out the survival rate, we had to find out the change of CFU, the number of bacteria is too large in protein expressed broth, since they are in the stationary phase, so we had to do gradient dilution.
And then spread the diluted broth to the petri dish, showed that when the broth was diluted to 10^13 times, we could count the cfu.
Desiccation
We used centrifuge machine and concentrator to remove the water.
Irradiation
More information, please go to our demonstration page.
