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the general public, who consumes poultry everyday. </div> | the general public, who consumes poultry everyday. </div> | ||
<div class="article-title">Supporting Experiment Results</div> | <div class="article-title">Supporting Experiment Results</div> | ||
− | <div class="article-content"><b>Secretory expression of pSIP403-PUS- | + | <div class="article-content"><b>Secretory expression of pSIP403-PUS-xynA in Lactobacillus reuteri</b></div> |
− | <div class="article-content">1. After the PCR identification sequence of pSIP 403-PUS-xynA | + | <div class="article-content">1. After the PCR identification sequence of pSIP 403-PUS-xynA bacteria liquid is |
correct, two parallel test groups are inoculated into 10ml of MRS liquid medium containing 5 ug/mL | 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;</div> | erythromycin, and cultured in a shaker at 37°C for 15 hours until the OD600 is 1.8722. And 2.2173;</div> | ||
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<div class="img-wrap no-margin"> | <div class="img-wrap no-margin"> | ||
<img src="https://static.igem.org/mediawiki/2021/6/6c/T--Shanghai_City_United--chg5.jpg" alt="" /> | <img src="https://static.igem.org/mediawiki/2021/6/6c/T--Shanghai_City_United--chg5.jpg" alt="" /> | ||
− | <span>OD600 of L. reueri/ pSIP403-PUS- | + | <span>OD600 of L. reueri/ pSIP403-PUS-xynA against induction time</span> |
</div> | </div> | ||
<div class="article-content"> | <div class="article-content"> | ||
− | From the experiment results above, the growth of pSIP403-PUS- | + | From the experiment results above, the growth of pSIP403-PUS-xynA reaches its optimal status at around |
24h. | 24h. | ||
</div> | </div> | ||
− | + | ||
− | + | ||
<div class="article-content"><b>DNS enzyme activity detection</b></div> | <div class="article-content"><b>DNS enzyme activity detection</b></div> | ||
<div class="article-content">1. Detection principle: 3,5-dinitrosalicylic acid solution and reducing sugar | <div class="article-content">1. Detection principle: 3,5-dinitrosalicylic acid solution and reducing sugar | ||
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ng/mL SppIP was induced for 8-12 hours. Is the best induction time. </div> | ng/mL SppIP was induced for 8-12 hours. Is the best induction time. </div> | ||
<div class="article-content">In summary, our experiment results fully indicate that we have successfully | <div class="article-content">In summary, our experiment results fully indicate that we have successfully | ||
− | obtained a probiotic, namely pSIP403-PUS- | + | obtained a probiotic, namely pSIP403-PUS-xynA in Lactobacillus reuteri, which is functional in secreting |
xylanase. </div> | xylanase. </div> | ||
<div class="article-content">Relying on this experiment results, we may further test the feasibility of our plan | <div class="article-content">Relying on this experiment results, we may further test the feasibility of our plan | ||
Line 193: | Line 192: | ||
<div class="img-wrap no-margin"> | <div class="img-wrap no-margin"> | ||
<img src="https://static.igem.org/mediawiki/2021/5/52/T--Shanghai_City_United--chg7.jpg" alt="" /> | <img src="https://static.igem.org/mediawiki/2021/5/52/T--Shanghai_City_United--chg7.jpg" alt="" /> | ||
− | <span>Average unit enzyme activity of L. reueri/ pSIP403-PUS- | + | <span>Average unit enzyme activity of L. reueri/ pSIP403-PUS-xynA against induction time</span> |
</div> | </div> | ||
</div> | </div> |
Latest revision as of 07:09, 20 October 2021
Proof of Concept
Overview
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.
Supporting Experiment Results
Secretory expression of pSIP403-PUS-xynA in Lactobacillus reuteri
1. After the PCR identification sequence of pSIP 403-PUS-xynA 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.
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
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 .
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.
Standard curve of glucose solution
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
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-xynA 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.
Average unit enzyme activity of L. reueri/ pSIP403-PUS-xynA against induction time