Team:Shanghai City United/Notebook
NOTEBOOK
8.16
Today we introduced ourselves and got familiar with each other, and through the
game named RUSH 60s we worked together for the first time to increase the exchange of topics and develop
team tacit understanding; our dry team coach met with us, analyzed the tasks in the training set, let us
understand the specific content of the tasks, and organized us to start the team name design.
8.17
We have laboratory safety training in the morning and configuration of LB medium,
autoclaving, PCR amplification of XynA gene in the afternoon. We also did agarose gel electrophoresis and
successful PCR, at last we cut the glue for backup.
8.18
Today, the xynA gene fragment was recovered from the gel, ligated with pMD19-T
vector, transformed into E. coli DH5a receptor cells, resuscitated, coated with LB/Amp+IPTG+X-Gal plates,
and incubated overnight at 37 degrees Celsius.
8.19
Some blue and white spots grew on LB/Amp+IPTG+X-Gal plates, and white single
colonies (i.e. E.coli/pMD19-T-xynA) were picked and incubated in LB/Amp liquid medium. We preserved
E.coli/pSIP403, then extracted plasmid pSIP403, followed by double digestion of plasmid pSIP403, PUS,
electrophoresis and gel cutting, respectively, where PUS was repeatedly done, double digestion overnight.
8.20
We draw plasmid pMD19-T-xynA today, double digestion identification and
electrophoresis. Just two of them are correct, we send sequencing identification, gum recovery pSIP403
double digestion product, PUS double digestion product, clean recovery xynA double digestion product.
8.21
Day-off
8.22
We collected the cell, ultrasonic crushing and high-speed centrifugation, protein
supernatant and protein precipitation were obtained, and SDS-PAGE protein glue was prepared. PSIP403, PUS
and xynA were connected by T4, transformed into DH5a, resuscitated, and coated with LB/ red mycin plate. The
correct sequencing plasmid PMD19-XYNA was transformed into BL21 (DE3), resuscitation, and coated with LB/Amp
plate. The correct sequencing strain DH5a/ PMD19-XYNA was grafted into 100mlLB, the OD was cultured to 0.4,
and IPTG was added to induce protein expression overnight.
8.23
We collected protein expression of bacteria from yesterday, ultrasonic broken,
low-temperature high speed centrifugal, protein and protein precipitation, numbered 1, 2, along with
yesterday for protein precipitation and number 3, with SDS-page gel electrophoresis, coomassie brilliant
blue staining rapidly and then quickly decoloring twice, third time decoloring slow and stay overnight. The
PUS fragment was amplified by PCR and new double restriction sites
EcoRI and NheI were introduced. Single colony BL21 (DE3)/PMD19-XYNA was isolated
into LB/Amp test tubes and cultured overnight.
8.24
We transferred BL21 (DE3)/PMD19-XYNA to 200mlLB medium with OD of 0.39 and added
IPTG to induce protein expression overnight. PCR amplified PUS fragments were recovered by electrophoresis
and gelled, and digested overnight with double enzymes. With double enzyme digestion of pSIP403 plasmid
overnight, we take photos for the protein glue and check the protein band of xynA, detected the enzyme
activity of xynA protein by DNS method.
8.25
Today, the members of the wet team collected thales, ultrasonic fragmentation,
centrifugation, prepared protein glue, protein electrophoresis, staining and decolorization; We then carried
out glue recovery/clean recovery of yesterday's double digestion product and finally achieved the connection
by T4 ligase.
8.26
Our team transformed the T4 ligatation products of yesterday's PUS, xynA and
pSIP403 into DH5a, revived and coated. The decolorized protein glue was photographed and xynA protein bands
were checked to confirm that xynA protein was expressed in the supernatant of the culture medium. DNS method
was used to detect the enzyme activity of xynA protein, which showed obvious brownish red color, and the
qualitative result was successful.
