Difference between revisions of "Team:Shanghai City United/Results"

Figure 1 is about the electrolysis of XynA. The result of electrolysis can clearly show whether the XynA is successfully amplified. XynA was amplified by PCR. The length of XynA is 650 bp. By compared the marker and the place of DNA, the PCR of XynA is successful.

Channel 1: pMD19-T-xynA-LCX-ZZY, correct

Channel 2: pMD19-T-xynA-HHJ, correct

Channel 3: pMD19-T-xynA-HX, incorrect

Channel 4: pMD19-T-xynA-LJL, incorrect

Channel 5: pMD19-T-xynA-0, incorrect

Channel 6: pMD19-T-xynA-SXY, incorrect

Channel 7: pMD19-T-xynA-CYF, incorrect

Channel 1~2: pSIP403, 6kbp, correct

Channel 3~4: PUS, 300bp, correct

After the sample has been centrifuged and sonicated. we put it to SDS-PAGE(SDS-polyacrylamide gel electrophoresis) which could determine the level of protein expression. As seen in the gel map (Maker, culture solution, intracellular supernatant, intracellular precipitation), the second red line of Marker represents 25KDa and the target protein should be 23.28kDa(Fig. 5). The blue line in the culture solution was near 23 KDa, it indicates that our protein could be sucessfully expressed in E. coli.

According to the sequencing results (Fig.6) from the sequencing company, the solid black line is the ideal state of PMD19-T-XynA and the DNA sequence of our plasmid matches the profile.

The enzyme activity test results showed groups from the left to the right, respectively: blank, culture medium supernatant * 2, intracellular supernatant * 2, and intracellular precipitation * 2. It could be seen by naked eyes that in the seven bottles of solution, the culture medium solution showed red after DNS reaction, which indicated that we had xylanase with well enzyme activity to degrade xylan and this result is consistent with the SDS PAGE result.

In Figure 8, it can be clearly seen that our plasmid pSIP403-PUS-xynA has been successfully transformed into Lactobacillus reuteri.

Secretory expression of pSIP403-PUS-xynA in Lactobacillus reuteri

The recombinant strain is added with 25 ng/mL SppIP to induce expression. After shaking at 37°C and 220r/ml for 3 hours, samples are collected at different time points to establish a function model of time and secreted expression.

From the experiment results above, the growth of pSIP403-PUS-xynA reaches its optimal status at around 24h.

Detection principle: 3,5-dinitrosalicylic acid solution and reducing sugar solution are reduced to brown-red amino compound after being heated together. Within a certain range, the amount of reducing sugar and the degree of brown-red substance color are reduced. In a certain proportional relationship, the absorbance of the resulting compound can be measured, and the concentration of reducing sugar can be deduced from the standard curve.

The DNS color method was used to detect the unit enzyme activity of two parallel group samples at different induction time points, and the average unit enzyme activity was calculated. The experimental data showed that the enzyme activity was maximum when 25 ng/mL SppIP was induced for 8-12 hours which would be the best induction time.

As our product is used as an additive for the poultry, safety would be the top priority we consider about. E. coli is mainly for experimental trial use which will not be considered as the final carrier of the xynA gene because it might have influences on disturbing the intestinal flora of the poultry. The ideal carrier would be Lactobacillus reuteri and we have constructed the recombinant L. reueri successfully which has also been proved to be capable of secreting xynA enzyme with good activity.
Next we will consult more experts for the product development advice and more tests would be necessary to ensure the stability and no side effect of our product. Currently, we have some thoughts about the plans of the future experiment:
1. Collect the hydrolyzed xylan to culture several bacteria, including the beneficial and the harmful so as to tests the influences on their growth, which is designed as a simulation of the effect of our product on the intestinal flora of the poultry.
2. The stability and the enzyme performance would be affected by the acidic conditions in the gastrointestinal mucosa of poultry thus we will take pH, temperature, etc. into account to further analyze the performance of both our engineered L. reuteri and the xynA enzyme.