Team:Shanghai Metro HS/Results
Results
Figure 1. The result of enzyme cutting and PCR
This picture is the nucleic acid electrophoresis result of enzyme cutting and
PCR(By performing PCR, we can obtain the desirable PKC-OP’s gene segment.). Column “marker” is a column that
is used to show the position of different lengths of genes. In this step, our aim is to verify whether these
results are the desired ones.
In figure 1, the number labeled above figure 1 represents different results. To begin with, number 1 and 2
is the result for pET-25b after enzyme cutting. Additionally, number 3 is the pET-25b without enzyme
cutting. At last, number 4 and 5 is the PKC-OP after PCR. As our experiment moves on, the data of our PKC-OP
and pET-25b are 233.2ng/μL and 17.8ng/μL. Lastly, we use the homologous combination to combine them.
Figure 2 E. coil having the desired pET-25b and PKC-OP
The pET25b-PKC-OP was constructed.
The plate shows monoclonals of pET25b-PKC-OP constructs
Figure 3. The result of Apa1 enzyme digestion validation
Column “marker” is a column that is used to show the position of different lengths of genes. Number 1 to 10
is the result for recombinant plasmid pET25b-PKC-OP after Apa1 enzyme digestion. We get two bands of 4712bp
and 1894bp. It further indicates that the obtained monoclonals were positive monoclonals containing the
recombinant plasmid.
Future plan: Testing the effectiveness of producing cellulase of positive
recombinant bacteria. This is important because we need to know how much bacteria is needed to be used to
effectively help animals digest fodder. In this experiment, we can control the time that positive
recombinant bacteria grow and use the OD test to set the numbers of bacteria. Then put different numbers of
bacteria and cellulose filter paper together. OD test can be used again to see the glucose concentration.
We designed several different induction conditions for protein expression. As the vector we chose to
construct the plasmid contains a pelB signal peptide, the engineered strain would secret the protein into
the medium. Therefore, we collected the culture supernatant after induction and ran SDS-PAGE for
verification.
According to the calculation by Snapgene, the expected protein molecular shall weigh 45.6 KDa. As seen from
the SDS-PAGE map (Fig. 4), there is a wide band just above 40 KDa and it indicates that we have obtained the
PKC-OP protein.
Future Plan
Testing the effectiveness of producing cellulase of positive recombinant bacteria.
This is important because we need to know how much bacteria is needed to be used to effectively help animals
digest fodder. In this experiment, we can control the time that positive recombinant bacteria grow and use
the OD test to set the numbers of bacteria. Then put different numbers of bacteria and cellulose filter
paper together. OD test can be used again to see the glucose concentration.