iGEM 2021 — Team Shanghai_City_United

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# Team:Shanghai City United/Notebook

Shanghai\_City\_United

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![](https://static.igem.org/mediawiki/2021/2/23/T--Shanghai_City_United--Description01.png)

NOTEBOOK

8.16

Today we introduced ourselves and got familiar with each other, and through the
game named RUSH 60s we worked together for the first time to increase the exchange of topics and develop
team tacit understanding; our dry team coach met with us, analyzed the tasks in the training set, let us
understand the specific content of the tasks, and organized us to start the team name design.

![](https://static.igem.org/mediawiki/2021/0/0f/T--Shanghai_City_United--Notebook01.jpg)

![](https://static.igem.org/mediawiki/2021/4/42/T--Shanghai_City_United--Notebook02.jpg)

8.17

We have laboratory safety training in the morning and configuration of LB medium,
autoclaving, PCR amplification of XynA gene in the afternoon. We also did agarose gel electrophoresis and
successful PCR, at last we cut the glue for backup.

![](https://static.igem.org/mediawiki/2021/d/dc/T--Shanghai_City_United--Notebook03.jpg)

8.18

Today, the xynA gene fragment was recovered from the gel, ligated with pMD19-T
vector, transformed into E. coli DH5a receptor cells, resuscitated, coated with LB/Amp+IPTG+X-Gal plates,
and incubated overnight at 37 degrees Celsius.

![](https://static.igem.org/mediawiki/2021/c/ce/T--Shanghai_City_United--Notebook04.jpg)

8.19

Some blue and white spots grew on LB/Amp+IPTG+X-Gal plates, and white single
colonies (i.e. E.coli/pMD19-T-xynA) were picked and incubated in LB/Amp liquid medium. We preserved
E.coli/pSIP403, then extracted plasmid pSIP403, followed by double digestion of plasmid pSIP403, PUS,
electrophoresis and gel cutting, respectively, where PUS was repeatedly done, double digestion overnight.

![](https://static.igem.org/mediawiki/2021/7/78/T--Shanghai_City_United--Notebook05.jpg)

![](https://static.igem.org/mediawiki/2021/f/f6/T--Shanghai_City_United--Notebook06.jpg)

8.20

We draw plasmid pMD19-T-xynA today, double digestion identification and
electrophoresis. Just two of them are correct, we send sequencing identification, gum recovery pSIP403
double digestion product, PUS double digestion product, clean recovery xynA double digestion product.

![](https://static.igem.org/mediawiki/2021/5/57/T--Shanghai_City_United--Notebook07.jpg)

![](https://static.igem.org/mediawiki/2021/4/4c/T--Shanghai_City_United--Notebook08.jpg)

8.21

Day-off

![](https://static.igem.org/mediawiki/2021/6/6f/T--Shanghai_City_United--Notebook09.jpg)

8.22

We collected the cell, ultrasonic crushing and high-speed centrifugation, protein
supernatant and protein precipitation were obtained, and SDS-PAGE protein glue was prepared. PSIP403, PUS
and xynA were connected by T4, transformed into DH5a, resuscitated, and coated with LB/ red mycin plate. The
correct sequencing plasmid PMD19-XYNA was transformed into BL21 (DE3), resuscitation, and coated with LB/Amp
plate. The correct sequencing strain DH5a/ PMD19-XYNA was grafted into 100mlLB, the OD was cultured to 0.4,
and IPTG was added to induce protein expression overnight.

![](https://static.igem.org/mediawiki/2021/d/d3/T--Shanghai_City_United--Notebook10.jpg)

8.23

We collected protein expression of bacteria from yesterday, ultrasonic broken,
low-temperature high speed centrifugal, protein and protein precipitation, numbered 1, 2, along with
yesterday for protein precipitation and number 3, with SDS-page gel electrophoresis, coomassie brilliant
blue staining rapidly and then quickly decoloring twice, third time decoloring slow and stay overnight. The
PUS fragment was amplified by PCR and new double restriction sites

EcoRI and NheI were introduced. Single colony BL21 (DE3)/PMD19-XYNA was isolated
into LB/Amp test tubes and cultured overnight.

![](https://static.igem.org/mediawiki/2021/b/b0/T--Shanghai_City_United--Notebook11.jpg)

8.24

We transferred BL21 (DE3)/PMD19-XYNA to 200mlLB medium with OD of 0.39 and added
IPTG to induce protein expression overnight. PCR amplified PUS fragments were recovered by electrophoresis
and gelled, and digested overnight with double enzymes. With double enzyme digestion of pSIP403 plasmid
overnight, we take photos for the protein glue and check the protein band of xynA, detected the enzyme
activity of xynA protein by DNS method.

![](https://static.igem.org/mediawiki/2021/d/d9/T--Shanghai_City_United--Notebook12.jpg)

8.25

Today, the members of the wet team collected thales, ultrasonic fragmentation,
centrifugation, prepared protein glue, protein electrophoresis, staining and decolorization; We then carried
out glue recovery/clean recovery of yesterday's double digestion product and finally achieved the connection
by T4 ligase.

![](https://static.igem.org/mediawiki/2021/e/e7/T--Shanghai_City_United--Notebook13.jpg)

8.26

Our team transformed the T4 ligatation products of yesterday's PUS, xynA and
pSIP403 into DH5a, revived and coated. The decolorized protein glue was photographed and xynA protein bands
were checked to confirm that xynA protein was expressed in the supernatant of the culture medium. DNS method
was used to detect the enzyme activity of xynA protein, which showed obvious brownish red color, and the
qualitative result was successful.

![](https://static.igem.org/mediawiki/2021/9/92/T--Shanghai_City_United--Notebook14.jpg)

Contact Info.

WeChat Official Account:*谷饲嘉Grain Fed Plus*

Email：*xxkk5905@gmail.com*

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Shanghai City United/Engineering

Shanghai\_City\_United

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Engineering

Background

Feed grains are whole grains such as corn, wheat and barley used in the feeding of
livestock and poultry. At present, corn is the main fodder in the feed industry. Due to the increasing
shortage of raw materials and the rising price, the development of the feed industry has been greatly
limited. One of the important measures to alleviate the shortages of corn is to fully develop wheat, grain,
and bran, which are abundant in China, to replace corn. However, the cell walls of cereals such as wheat,
cereal, and bran contain anti-nutritional factors such as arabinoxylan and non-starch polysaccharide (NSP),
which will affect the digestibility of nutrients in single-stomach animals and the absorption of nutrients
in poultry. Arabinoxylan is a polysaccharide found in rice bran (hemicellulose B) edible fiber. Therefore,
we decide to aim at adding xylanase to the feed to degrade arabinoxylan, so as to improve the feed
absorption efficiency.

Design

Our project is to design an edible "drink" for monogastric animals, mainly poultry
through biosynthesis technology. The core product will be a probiotic that can produce xylanase. In this
project, we designed to construct a plasmid expressing xylanase gene, then we transformed them into
Escherichia coli and Lactobacillus reuteri for further performance analysis.

Compared to traditional feed, our drink contains the probiotics that could help
poultry digest xylan, thus
increasing feed efficiency. In this way, not only can the time and economic cost of feeding be saved, but
also the gastrointestinal tract of the animal can be protected and thus the disease rate can be reduced.

Build

Plasmids pMD19-T-xynA and pSIP403-PUS-xynA were constructed to produce
recombinant
xylanase in E. coli and L. reuteri, respectively (Figure 1).

![](https://static.igem.org/mediawiki/2021/8/88/T--Shanghai_City_United--chg8.jpg)
Figure 1. Schematic maps of pMD19-T-xynA and pSIP403-PUS-xynA.

Test

pMD19-T-xynA was transformed into E. coli BL21(DE3). After the sample has been
centrifuged and sonicated.
The level of protein expression was determined using SDS-PAGE. As seen in the gel (Maker, culture solution,
intracellular supernatant, intracellular precipitation), the second red line of Marker represents 25KDa and
the target protein should be 23.28kDa (Figure 2). The blue line in the culture solution was near 23 KDa, it
indicates that our protein could be successfully expressed in E. coli (Figure 2).

![](https://static.igem.org/mediawiki/2021/d/dc/T--Shanghai_City_United--engineering02.jpg)
Figure 2. SDS-PAGE result of BL21(DE3)/pMD19-xynA.

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.

![](https://static.igem.org/mediawiki/2021/0/06/T--Shanghai_City_United--engineering03.jpg)

Figure 3. The xylanase activity was tested by DNS method. 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.

Plasmid pSIP403-PUS-xynA transformed into L. reuteri to for secretory expression of
xynA in L. reuteri. As
shown in Table 1, significant xylanase activity was detected from broken supernatant by DNA method.

Table 1. Xylanase activity (OD540 nm) was detected from broken supernatant by DNA
method.

![](https://static.igem.org/mediawiki/2021/a/a7/T--Shanghai_City_United--engineering04.jpg)

A standard curve for reducing sugar was prepared using glucose (Figure 4).

![](https://static.igem.org/mediawiki/2021/c/c4/T--Shanghai_City_United--chg6.jpg)
Figure 4. Standard curve for reducing sugar.

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 inducer was added for 8-12 hours
(Table 2).

Table 2. Calculation of average unit enzyme activity.

![](https://static.igem.org/mediawiki/2021/1/11/T--Shanghai_City_United--engineering06.jpg)

Contact Info.

WeChat Official Account:*谷饲嘉Grain Fed Plus*

Email：*xxkk5905@gmail.com*

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# Team:Shanghai City United/Experiments

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![](https://static.igem.org/mediawiki/2021/2/23/T--Shanghai_City_United--Description01.png)

EXPERIMENTS

1. Preparation of LB medium

Materials:

Yeast extract 5g/L

Tryptone 10g/L

NaCl 10g/L

Agar(only for solid medium)1.5%

H2O (as solvent) 1000ml

Totally 1000ml (600ml for liquid medium, 400ml for solid
medium)

Steps:

1. Weighing 5 gram yeast extract

2. Preparing 1000ml, and pouring into a huge measuring
cylinder

3. Adding the yeast extract into water

4. Weighing 10 gram Tryptone

5. Adding the tryptone into water

6. Weighing 10 gram sodium chlorine

7. Adding sodium chlorine into water

8. Stirring the solution

9. Preparing 5 flasks 3 of them are 250ml, and 2 are 500ml

10. Adding 150ml solution into the 250ml flasks, and adding
300ml solution into 500ml solution

11. For solid medium, adding the agar into the bottom of
the flask first, and than pouring the solution into it.

12. Use the membrane to seal the flask( double filter, 4
pores for air recycle)

13. Put the flask into the High-Pressure Steam
Sterilization Pot for sterilization(0 is standard pressure 1atm)(condition: 0-0.4, 120 centigrade 30 minute)

2. PCR amplification

Materials:

Primer-F 1μL

Primer-R 1μL

2X Prime Star Max(DNA polymerase ,Buffer, dNTP) 50μL
dillute to 1/2 25μL

Template 1μL

ddH2O 22μL

In the PCR machine:

98℃ 5m pre-denaturation (break the hydrogen bond)

98℃ 15s break the hydrogen bond

55℃ 15s primer connect to the brands

72℃ 5s/1k bp extend the DNA strands(the temperature which
the enzyme most active)

Repeat these three steps 30 times

72℃ 3m let the DNA strands completely extend sufficiently

4℃ preserve the DNA

Process:

1. Primer 5.00 n mol diluter in water

2. 500μL water add into Primer-F

3. Split 200μLPrimer-F

4. Adding template into 900μLwater(dilute)

5. Adding 900μLwater into template

6. Adding 22μL ddH2O into the PCR tube

7. Adding 25μL Primer Star Max into the PCR tube

8. Adding 2 primers

9. Adding template

10. Marked the number (determine)

11. Put in the PCR machine(shut the top cover avoid the
liquid evaporate)

12. Setting the program has written above.

13. Waiting for the process finish

14. Preserve the DNA.

3. Running nucleic acid electrophoresis

Mechanism: DNA is negative charge, so it will be attract to the positive
direction. Also, different DNA contains different number of base pair and shape. The speed of DNA are
different either. We can compare the model DNA(500bp) with ours product after PCR to determine whether our
products are that one we need.

A. Nucleic Acid Electrophoresis Gel

Materials:

1. Agarose 1%

2. TAE buffer 100ml

3. Nucleic acid dye 10μL

4. Marker

5. DNA from PCR

Process:

1. Mix the agarose and 100ml TAE buffer

2. Heated in microwave about 3 min until all the agarose
melt.

3. Add Nucleic acid dye into the solution.(double glove
avoid the dye contact skin)

4. Pour the heated solution into the mold tank and plate
and wait it to solid

5. Take the gel(use knife around the gel and pick it up)

6. Put the gel in the bottom of the tank

7. Pour TAE buffer into the tank until it cover the gel

8. Add the marker into the first ole of the gel(do not
penetrate the gel)

9. Add Loading buffer into PCR tube(let the DNA get color)

10. Add other DNA into the gel.

11. Clean the surrounding of the tank and cover it

12. Energized it with 120V(there are bubbles going out from
the liquid)

13. Waiting about 15 min

14. Observe the traits of the DNA under the violet light.

4. Measuring OD600

Mechanism: The light absorption of bacteria is used to measure the concentration of
bacterial culture medium, so as to estimate the growth of bacteria. It is usually used to refer to the
density of bacterial cells.
Experimental Procedure

1. To measure the concentration, we use Nanodrop by
measuring light absorption value.

2. Use 2ul Micro pipette with ddH2O to clean the water on
the sampling table, wipe the sweat with dust-free paper, repeat three times.

3. Drop 2ul DNA sample on the detection platform and the
internal light penetrate the DNA sample.( We have 100% internal light, if the metering wall shows 20% light,
then we will know that the DNA sample absorb 80% light.) (A260/A230→calculation of concentration,
A260/A280→calculation of purity—1.8 is close to pure)

4. After the experiment, the ddH2O cleaning process should
be repeated three times again.

5. Building Plasmid (Double Digest&T4 connection)

Lactobacillus’s route:

1. Carry on processing the previous E. coli that carries
XynA, select white strains that express lac ab enzyme.

2. Injecting the selected strains into LB nutrient solution
and Amp antibiotic solution.

3. Extracting the plasmids from PMD19-T—XynA. (Which has
been mentioned in detail later)

4. Double digestion for different biological parts that
eventually constructed the final plasmid:

First time:

Using restriction enzymes to cut
different parts that serves for the connections. Using NheI and HindIII to cut the part of XynA; Using BamHI
and NheI to cut the part of PUS; Using BamHI and HindIII to cut the part of PSIP403. Three cutted parts need
to run the nucleic acid electrophoresis, getting the target length around 500bp for XynA; 300bp for PUS and
6kbp of PSIP403.
(Notice worthily, by using the formal strategies on double digestion, we failed on the step of transplanting
the building plasmids into the Lactobacillus. Excluding the effect of erythromycin, we’ve concluded that two
restriction sites on the parts may cut off the segment of the genetic expression for ori---a codon initiator
for the functioning of this plasmid inside the Lactobacillus.)

Second time:

By first using PCR techniques for
amplifying the restriction site fragment, we’ve changed one of our restriction enzymes from BamHI to EcoRI.
(Details for the solutions are provided bellow)

![](https://static.igem.org/mediawiki/2021/9/94/T--Shanghai_City_United--Experiment01.jpg)

5. T4 connecting system

(PSIP403：PUS : XynA=1:1:1)

10X T4 Ligase buffer 1μL

psiP403 0.5μL

Pus 5.5μL

XynA 2μL

T4Ligase 1μL

Solutions above need to be mixed in a testing tube to react for at least 30 minutes
under the surrounding temperature of 6 degrees.

6. Cultivating E.coli

Lab procedure:

1. Process the E.coli DH5α with Cacl2 to depolarize the
membrane of the cell and allow access of exterior DNA.

2. Extract 50ul of E.coli DH5α from stored glycerin. And
transfer into a 2ml Eppendorf tube.

3. Extract and add 5ul of T4 Polymerase (XynA) to the
Eppendorf tube.

4. Unfreeze the minus eighty degrees’ cells on ice for 30
minutes. (In order to allow DNAs to do free diffuse)

5. Water bath the Eppendorf tube to heat shock the E.coli
for 90 seconds to let the plasmids absorb through the openings on the cell.

6. Place the Eppendorf tube on ice for 30 minutes to
entirely close the openings on the cell and recovering the cell from competent.

7. 895ul of LB liquid culture medium were added under the
sterile operating table as nutrients.

8. The Eppendorf tube then put in a beaker and placed in a
shaking incubator for 30 minutes. (240rpm)

9. Then the Eppendorf tube was centrifuged for 30 seconds.
(12000 times/min, centrifugal force: 13800)

10. Solid LB culture medium was added into petri dish with
a final concentration of 100ug/ml Amp filtrated stock; 20ug/ml IPTG filtrated stock and 20ug/ml X-Gel
filtrated stock.

11. Burned the micro pipette tips with the alcohol burner
and placed upside down till the top of tip smoothen.

12. Extract 800ul of bacteria from the Eppendorf tube and
safe the rest of it.

13. 50ul of droplets should gently drop in the center of
the petri dish and rub back and forth to coated the plate.

14. Put the coated plate upside down into the incubator and
incubate overnight at 37℃.

7.Plasmid extraction（PMD19-XynA)

1. Add E.coli DH5 α/PSIP403-PUS-XynA into 4ml LB solution
and put it into shaking incubator. (37℃, 240rmp, 16h)

2. Add 250ul buffer SP1 and blow and mix well. (SP1:
Glucose- form a suspension with suspended bacteria; RNase- degrade RNA; Buffer- provide the environment)

3. Add 250ul buffer SP2, reverse it 5 to 10 times. SP2:
NaOH–Cleavage cell wall, slightly clarified liquid, DNA&RNA release from the cell; metal chelating
agent–wipe off the metal ion

4. Add 350ul buffer SP3, quickly invert and mix. SP3: SDS
(protein denaturant)–Let the protein denature; CH3COOK–Neutralize PH (→NaOH), K→ The metal ions are turned
into water-insoluble salts and precipitated

5. Centrifuged it in a centrifuge. (5min, 12000 times/min,
centrifugal force: 13800) Then we get: Sediment: Protein, metal ions (some of), genome (wrap around the
protein)/Supernatant liquid: plasmid DNA, metal ions (some of ), residual protein (some dissolve in the
water)

6. Add 500ul buffer S on the adsorption column. Centrifuged
it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800) Pour out the waste liquid at the bottom.
(Column can be activated and balanced./ Wash the silicate fiber membrane.)

7. Add 800ul supernatant liquid on the adsorption column.
Centrifuged it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800) Pour out the waste liquid at
the bottom. (Plasmid DNA can be attached to the membrane.)

8. Add 500ul buffer DW1 on the adsorption column.
Centrifuged it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800)Pour out the waste liquid at
the bottom. (Remove residual protein)

9. [Add 500ul wash buffer(C2H6O) on the adsorption column.
Centrifuged it in a centrifuge (30s, 12000 times/min, centrifugal force: 13800) Pour out the waste liquid at
the bottom.] \*2 （Cleaning residue, like salt, metal, ions...)

10. Put it in a centrifuge without adding any liquid. (30s,
12000 times/min, centrifugal force: 13800) (Centrifuge the wash buffer)

11. Move the column to a new 1.5ml tube, open the lid and
let stand for 1min. (C2H6O volatulization)

12. Add 50ul ddH2O (50℃ hot water) (Elution buffer cannot
be used, Elution buffer--Tris-HCl×) Centrifuged it in a centrifuge (60s, 12000 times/min, centrifugal force:
13800) (Eluting plasmid DNA)

13. Throw the column into the lab pollution bin.
(Remaining: plasmid DNA and ddH2O)

8. Running SDS PAGE protein electrophoresis

Make SDS PAGE gel

1. 2.7ml lower layer solution, 2.7ml lower layer buffer,
60ul improved coagulant. (Blow and mix. It will set in 5-10 minutes)

2. Slot: Add water and see if the liquid level drops. It is
set when the liquid level doesn’t drop.

3. Add 1000ul for each time (The distance between the
liquid level and the short glass plate is 0.5cm longer than the comb length.)

4. Add some water to see if the liquid is set.

5. 0.75ml upper glue solution, 0.75ml red upper buffer,
15ul coagulant, mix, pour in, and insert the comb.

Running

Mechanism: Spacer gel: superstratum, small concentration, large pore. It helps the
protein to press into a
line at the boundary between the upper and lower layers.
Separation gel: substratum, large concentration, small pore. From here on, the small ones swim faster than
the big ones.

1. Power up and start running（120v/30min)

2. We can stop when the blue line reaches ths bottom.

Coomassie brilliant blue staining

1. Pour CBB into a box to cover the gel in it. Rapid
dyeing: microwave on medium for 1 minute. Shaking with hands for 3 minutes. Pour CBB.

2. [Pour CBB distaining solution. Rapid wash the dye:
microwave on medium for 1 minute. Shaking with hands for 3 minutes. Pour CBB distaining solution.] \*3-4

9. Measuring the enzyme activity

Xylan Liquid (50mL):

0.25g of xylan (5g/L)

Add ddH2O until 50mL

Reaction Medium (1mL):

![](https://static.igem.org/mediawiki/2021/1/1a/T--Shanghai_City_United--Experiment02.jpg)

·Water bath--37℃--15min, 90℃--5min, add DNS--2ml, 90℃--8min, add ddH2O 7ml (the
capacity to 10mL)

·Let it cool to room temperature.

·Measuring 540nm light absorption value（The procedure is the same as that for
OD600）

Contact Info.

WeChat Official Account:*谷饲嘉Grain Fed Plus*

Email：*xxkk5905@gmail.com*

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:Shanghai City United/Results

Shanghai\_City\_United

* [Home](https://2021.igem.org/Team%3AShanghai_City_United)
* Project

  + [Description](https://2021.igem.org/Team%3AShanghai_City_United/Description)
  + [Experiments](https://2021.igem.org/Team%3AShanghai_City_United/Experiments)
  + Results
  + [Proof Of Concept](https://2021.igem.org/Team%3AShanghai_City_United/Proof_Of_Concept)
  + [Notebook](https://2021.igem.org/Team%3AShanghai_City_United/Notebook)
  + [Safety](https://2021.igem.org/Team%3AShanghai_City_United/Safety)
* Parts

  + [Parts
    Collection](https://2021.igem.org/Team%3AShanghai_City_United/Parts)
  + [Engineering](https://2021.igem.org/Team%3AShanghai_City_United/Engineering)
  + [Contribution](https://2021.igem.org/Team%3AShanghai_City_United/Contribution)
* Human Practices

  + [Integrated Human Practice](https://2021.igem.org/Team%3AShanghai_City_United/Human_Practices)
  + [Communication](https://2021.igem.org/Team%3AShanghai_City_United/Communication)
  + [Fundraising](https://2021.igem.org/Team%3AShanghai_City_United/Fundraising)
* [Implementation](https://2021.igem.org/Team%3AShanghai_City_United/Implementation)
* [Entrepreneurship](https://2021.igem.org/Team%3AShanghai_City_United/Entrepreneurship)
* [Model](https://2021.igem.org/Team%3AShanghai_City_United/Model)
* Team

  + [Team
    Members](https://2021.igem.org/Team%3AShanghai_City_United/Members)
  + [Attributions](https://2021.igem.org/Team%3AShanghai_City_United/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AShanghai_City_United/Collaborations)
  + [Partnership](https://2021.igem.org/Team%3AShanghai_City_United/Partnership)

![](https://static.igem.org/mediawiki/2021/2/23/T--Shanghai_City_United--Description01.png)

Results

![](https://static.igem.org/mediawiki/2021/7/70/T--Shanghai_City_United--Results1.jpg)
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.

![](https://static.igem.org/mediawiki/2021/9/90/T--Shanghai_City_United--Results2.jpg)
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

![](https://static.igem.org/mediawiki/2021/0/0b/T--Shanghai_City_United--Results3.jpg)
Figure 3. electrophoregram of enzyme-digested products of pSIP403 and PUS

Channel 1~2: pSIP403, 6kbp, correct

Channel 3~4: PUS, 300bp, correct

![](https://static.igem.org/mediawiki/2021/f/f3/T--Shanghai_City_United--Results4.jpg)
Figure 4. SDS PAGE result of BL21(DE3)/pMD19-xynA, Coomassie blue staining.

![](https://static.igem.org/mediawiki/2021/e/e4/T--Shanghai_City_United--Results5-1.png)
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.

![](https://static.igem.org/mediawiki/2021/1/10/T--Shanghai_City_United--Results5.jpg)
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.

![](https://static.igem.org/mediawiki/2021/d/d8/T--Shanghai_City_United--Results6.jpg)
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.

![](https://static.igem.org/mediawiki/2021/1/1d/T--Shanghai_City_United--Results7.jpg)
![](https://static.igem.org/mediawiki/2021/8/88/T--Shanghai_City_United--Results8.jpg)
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.

![](https://static.igem.org/mediawiki/2021/7/75/T--Shanghai_City_United--chg-3.jpg)
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.

![](https://static.igem.org/mediawiki/2021/2/2c/T--Shanghai_City_United--chg4.jpg)
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.

Contact Info.

WeChat Official Account:*谷饲嘉Grain Fed Plus*

Email：*xxkk5905@gmail.com*