Difference between revisions of "Team:Shanghai City United/Proof Of Concept"

Monogastric animals can not digest the xylan contained in grain feed, which may lead to a series of digestion problems for monogastric animals. Poultry are typical monogastric animals. Poultry breeding industry impact our daily life, thereafter, how to provide poultry breeders with high-quality feed is a critical issue. To address this issue, we started our project, aiming at developing a probiotic (Lactobacillus) containing xylanase to produce feed additives and poultry beverages.

The application of our additives in grain feeds will then enhance poultry’s digestion. We believe that our additives will be a good choice for feed producers, poultry breeders and even the general public, who consumes poultry everyday.

1. After the PCR identification sequence of pSIP 403-PUS-xynA AM bacteria liquid is correct, two parallel test groups are inoculated into 10ml of MRS liquid medium containing 5 ug/mL erythromycin, and cultured in a shaker at 37°C for 15 hours until the OD600 is 1.8722. And 2.2173;

2. Then transfer 1ml to 200ml MRS liquid containing 5 ug/mL erythromycin, and continue to cultivate until the OD600 is 0.5304 and 0.6718;

3. At this time, start induction. 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;

4. Take 20ml of the recombinant strain after induction culture, of which 1ml is used to detect the OD600 of the sample with an ultraviolet spectrophotometer, the remaining 19ml is centrifuged, washed twice with 1 mL of 0.1 mol/L PBS (pH 6.0), and the pellet is resuspended in 2ml of PBS and ultrasonic broken.

5. Take the broken supernatant for DNS enzyme activity detection.

1. 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.

2. Configuration of reagents

(1) Commercial DNS reagents

(2) Glucose standard solution (1mg/ml): accurately weigh 0.1mg of anhydrous glucose, and dilute to a 100ml volumetric flask after dissolving; glucose standard solution (0.1mg/ml): take 10ml of the above 10-2mol/l The solution is diluted to a 100ml volumetric flask; according to the above dilution rule, the solution is diluted to different concentrations of glucose (shown in the table below).

3. Xylanase extraction: Centrifugal supernatants of broken samples at different induction time points;

4. Glucose standard curve drawing: take 1ml glucose standard solution into a 15ml centrifuge tube, add 2ml DNS reagent, boil water bath for 5 minutes, cool tap water to room temperature, take 1ml into a cuvette, and measure the OD value at 540nm. Take the absorbance value as the ordinate and each standard concentration (mg/ml) as the abscissa to obtain the glucose standard curve.

5.Sample detection DNS: Take 1ml of the broken bacteria induced at different time points and add it to a 15ml centrifuge tube, add 2ml DNS reagent, boil water bath for 5min, cool tap water to room temperature, take 1ml into a cuvette, and measure the OD value at 540nm .

Calculation of Unit Enzyme Activity, Enzyme Activity

n=Dilution Times
K: Curve Slope
T: Reaction Time, min
1000: mg is converted to ug

From the experiment results above, 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. Is the best induction time.

In summary, our experiment results fully indicate that we have successfully obtained a probiotic, namely pSIP403-PUS-xyn AM in Lactobacillus reuteri, which is functional in secreting xylanase.

Relying on this experiment results, we may further test the feasibility of our plan to make feed additives and poultry beverages.