Team:Shanghai City United/Results
Results
Figure 1. electrophoregram of XynA
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.
Figure 2. electrophoregram of pMD19-T-xynA enzyme-digested product
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
Figure 3. electrophoregram of enzyme-digested products of pSIP403 and PUS
Channel 1~2: pSIP403, 6kbp, correct
Channel 3~4: PUS, 300bp, correct
Figure 4. SDS PAGE result of BL21(DE3)/pMD19-xynA, Coomassie blue staining.
Figure 5. Protein molecular weight result of XynA by Snapgene
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.
Figure 6. sequencing result of PMD19-T-XynA
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.
Figure 7. DNS test results of E. coli/pMD19-xynA
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.
Figure 8. Colony PCR results of Lactobacillus reuteri/pSIP403-PUS-xynA and marker maps
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.
Figure 9. OD600 of L. reueri/ pSIP403-PUS-xynA against induction time
From the experiment results above, the growth of pSIP403-PUS-xynA reaches its
optimal status at around 24h.
DNS enzyme activity detection
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.
Figure 11. Average unit enzyme activity of L. reueri/pSIP403-PUS-xynA against induction time
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.
Concern and Future Plan:
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.
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.