iGEM 2022 — Team shanghai-united

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Notebook

2022.4.16

• The background, history, and scale of iGEM.
• Rules and where to find more information
• The products of some past gold-medalist teams
• A proposed timeline of our team’s progress

2022.4.23

• Eukaryotes, prokaryotes, their differences and similarities
• Basic information about DNA, RNA, and proteins
• The central dogma
• Basics of genetic engineering
• What GMOs are, and their benefits

2022.4.30

• Definition and features of market research.
• Macroµ market research
• Questionnaire design
• Analysis of collected data

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-notebook01.jpg)

• cloning and restriction enzymes.
• History of DNA structure, restriction enzymes, cloning, and Genentech.
• Watson and Crick’s discovery of the structure of DNA.
• Plasmids and some fundamentals of molecular biology.

2022.5.14

• Tips & preparation for expert interviews.
• What to do during an interview.
• How to check information after an interview.

2022.5.20

• Basic lab techniques
• Culture methods and procedures
• Structure and features of plasmids
• Protein expression and purification

2022.5.27

• Activity planning and publicity for popular science education
• How to prepare materials for the iGEM competition

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-notebook02.jpg)

2022.6.2

• What genes are and how they work
• More basic information about genes
• Biobricks and how to design or assemble them.

2022.6.9

• Business planning and strategies
• How to improve our products from a financial perspective
• agenda, commercialization, implementation and entrepreneurship

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-notebook03.jpg)

2022.6.18

• Explanation of modeling method and diagram drawing
• Components of a team for iGEM
• Requirements for awards

2022.7.18

• Learn about laboratory safety and how to use mechanical pipettes
• Prepare LB liquid and solid culture media
• Lean the principles of molecular cloning
• Use PCR to amplify PKC gene sequence

2022.7.19

• Use restriction enzymes to isolate target gene sequences from their original vectors
• Use gel electrophoresis to isolate gene sequences of the correct length
• Retrieve genetic material from the gel

2022.7.20

• Transform TOP10 competent cells with pET-28a, XynA, PKC, and CcxynA.
• Amplify density of pET-28a, XynA, PKC, and CcxynA

2022.7.21

• Learn about plasmid extraction
• Extract plasmids from TOP10 competent cells
• Pick monoclonal colonies of bacteria and inoculate in liquid medium
• Prepare liquid LB culture medium for further experiments

2022.7.22

• Expand the culture of bacteria
• Test density of bacterial culture (detect OD value)
• add IPTG to induce production of target proteins
• Prepare buffers for extraction of proteins

2022.7.23

• Extract and purify proteins from BL21 cells using ultrasonic lysis and Ni-NTA beads
• Test density of proteins in byproduct liquids to ensure all proteins have been extracted

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-notebook04.jpg)

2022.7.24

• Prepare gel for SDS-PAGE (including Coomassie brilliant blue staining solution)
• SDS-PAGE
• Observe results

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-notebook05.png)

2022.7.25

• Determination of the concentration of reducing sugar xylose with DNS and obtaining the standard curve
• Determination of enzymatic activity of four enzyme solutions ( xynA, PKC ,xyl3A and ccxynA )

2022.7.26

• Determination of enzymatic activity ( xynA, PKC ,xyl3A and ccxynA )
• Experimental data analysis
• Writing experimental results

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Engineering

Introduction

With the continuous expansion of the animal husbandry production scale, the demand for high-quality feed is also
increasing greatly, which makes the development of animal production face severe challenges of supply and demand of
feed resources. Therefore, it is urgent to find a way for improving feed efficiency. Cereal energy feed has high
potential nutritional value as feed for poultry. However, grain feed contains high soluble non-starch
polysaccharides, which hinders the utilization and absorption of poultry. Silage contains a large amount of
cellulose, which is the main structural component of the plant cell wall. It is usually combined with hemicellulose,
pectin, and lignin, and is difficult to digest and absorb. To solve this bottleneck problem, cellulase hydrolysis
has low efficiency and high cost. We adopt the strategy of multi-enzyme synergistic degradation, which can promote
the bioconversion of lignocellulose with cellulase. We further added the xylanase xyl3A gene to the vector, so that
it can be combined with the cellulase PKC-01 and simultaneously express the β-xylosidase to improve the utilization
rate of feed and the applicable varieties of livestock.

Design

Due to the T7 promoter and T7 RNA polymerase having strong ability in translation and usually being used as protein
expression, we choose pET28a-vector and E.coli BL21(DE3), to express our target protein xyl3A. Moreover, we inserted
the DNA sequences of xyl3A into the NheI and HindIII sites of the pET-28a vector (Figure 1.) and transferred the
plasmid into E.coli BL21(DE3) for protein expression.

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-engineering01.jpg)
Figure 1. The map of recombinant plasmid pET28a-xyl3A.

Build

In order to build our plasmids, we let the company synthesize the xyl3A integrate it into the pET28a vector. We
transform the plasmid pET28a-xyl3A into E.coli DH5α. Figure 2 showed that the plasmid pET28a-xyl3A was successfully
transform.

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-engineering02.jpg)
Figure 2. The PCR identification result of pET28a-ccxynA, pET28a-PKC, pET28a-xynA and pET28a-xyl3A

We send the plasmid pET28a-xyl3A to biological company for sequencing. The returned sequencing comparison results
showed that there were no mutations in the ORF region (Figure 3.)

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-engineering03.jpg)
Figure 3. The sequencing blast results of xyl3A

Next, the plasmid pET28a- xyl3A was extracted from E.coil DH5α, then transformed into
E.coil BL21(DE3). The PCR identification results showed that the transformation of plasmid pET28a-xyl3A was
successful (Figure 2).

Test

1.Protein expression and purification

We added IPTG to induce protein expression when the OD600 reached 0.3-0.5. After overnight induction and culture, we
collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins xyl3A. Next, we used
nickel column purification to purify the protein xyl3A we wanted.

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-engineering04.jpg)

Figure 4. The SDS-PAGE result of the four proteins
M:180KD marker
S：Soluble lysate
P：Precipitation
E：Elution

The molecular weights of xyl3A were 84.93KD, referring to the marker in Figure 4, we found the proteins xyl3A in
lane S, indicating that proteins were successfully expressed in E. coli BL21 (DE3).

2.Determination and analysis of enzyme activity

Determination of reducing the sugar by DNS method：The absorbance OD540 value of the purified enzyme solutions
(xyl3A) was measured after color reaction with DNS. The activity of the enzyme can be converted by the amount of
sugar consumed and the working time.

Table 1. The enzyme activity of xyl3A

| Four enzyme solutions | Enzyme activity(U/mL) |
| --- | --- |
| xyl3A | 0.302713345 |

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-engineering05.jpg)
Figure 5. The enzyme activity of xyl3A

The enzyme activity of xyl3A is 0.3U/mL. Figure 5 indicated that protein xyl3A was
successfully expressed, and
the enzyme activity of xyl3A was active. It indicated that the xyl3A has the function of decomposing xylan.

Learn

Our results showed that the pET28a-xyl3A plasmid was successfully constructed, and the proteins xyl3A were
successfully expressed in E.coli BL21 ( DE3). Meanwhile， the results indicated that the xyl3A has the function of
decomposing xylan. We can add an appropriate amount of xyl3A enzyme to feed to improve the efficiency of xylan
decomposition. We can adopt the strategy of multi-enzyme synergistic degradation to accelerate the degradation rate
of cellulose or xylan in feed, which can be added to feed as a feed additive to improve the ability of animals or
digest and utilize feed, promote animal appetite and improve the quality of animal husbandry products. It can solve
the bottleneck problem of low efficiency and high cost of cellulase hydrolysis. In the future, we can put into
large-scale use in animal husbandry, such as farms, and feed processing plants.

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Experiments

1. Polymerase Chain Reaction (PCR)

1.1 Preparing Fanta solution

• Combine all components:

| Reagent | volume | Concentration |
| --- | --- | --- |
| DNA sequence (template) | 1μl |  |
| primer-f | 1μl | 10μmol/ml |
| primer-r | 1μl | 10μmol/ml |
| DNA Polymerase | 25μl |  |
| double distilled water(ddwater) | 22μl |  |
| Total | 50ml |  |

1.2 Starting the PCR reaction

• Place the solution into PCR machine
• Start machine with following settings:

| Step # | Temperature (°C) | Time | Purpose |
| --- | --- | --- | --- |
| 1 | 95 | 3min | Break hydrogen bonds between nucleotides |
| 2 | 95 | 30s | Activate DNA polymerase |
| 3 | 55 | 30s | Primers attach to sense & antisense strands |
| 4 | 72 | 3min | DNA polymerase builds a complimentary strand |
| 5 | Repeat steps 2-4 25 times | | |
| 6 | 72 | 5min | Stabilize DNA |
| 7 | 12 | - | Storage |

2. Plasmid Construction

2.1 Preparing solutions for restriction enzymes.

• For pMD19-xynA (xynA) plasmid:

| For pMD19-xynA (xynA) plasmid | | For pUC57-PKC-OP (PKC) plasmid | |
| --- | --- | --- | --- |
| Reagent | Volume (μl) | Reagent | Volume (μl) |
| Pet28 a plasmid | 5 | Pet28 a plasmid | 5 |
| pMD19-xynA plasmid | 5 | PKC plasmid | 5 |
| Nhe I | 1 | Xba I | 1 |
| Hind III | 1 | BamH I | 1 |
| ddwater | 42 | ddwater | 42 |
| Cut smart | 6 | Cut smart | 6 |
| Total | 60 | Total | 60 |

2.2 Preparing agarose regular gel for gel electrophoresis

• Add 0.4g Agarose Regular to 40ml TAE Buffer
• Mix together and bring to a boil in a microwave (stop immediately once boiling)
• When the solution is cool enough to touch, remove from microwave and add 5μl GelRed
• Cool the solution away from light.
• Pour into gel electrophoresis mold with the comb inserted.
• Remove when solidified and place in gel electrophoresis machine. The end with indents left by the comb should be
facing the cathode.

2.3 Screening of target genes (gel electrophoresis and retrieving genes)

• Add 20μl PCR products, enzymatic plasmid cutting products, and marker (control group) into individual indents in
the agarose gel.
• Conduct electrophoresis at 140V for 30 minutes.
• Observe the gel under blue light. Bands of DNA show up as brightly lit.
• Slice the desired DNA fragment from the gel.
• Place the gel in a labeled microfuge tube.
• Add 650μl gel dissolving buffer (adjust amounts according to instructions on the kit) to the tube and immerse in
56˚C water bath.
• Transfer solution to adsorption columns and centrifuge at 5100g for 5 minutes, 4 times (add 650μl Wash Buffer in
2-3 times)

2.4 Enzymatic plasmid connecting

• Mix the solution and let it sit for 1 hour in 16°C.

| Reagent | Volume (μl) |
| --- | --- |
| Pet28 a plasmid | 11.4 |
| enzymatic plasmid cutting products (xynA) | 0.6 |
| T4 ligase | 1 |
| T4 ligase buffer | 2 |
| ddH2O | 5 |
| Total | 20 |

2.5 Homologous recombination

• For Xyloglucosidase (Xyl3A) and Acetylxylan esterase gene (CcXynA)
• Mix the solution and wait 30min in 37°C

| Reagent | Volume (μl) |
| --- | --- |
| Mix Buffer (5x) | 2 |
| Homologous recombinase | 1 |
| Pet28 a plasmid | 0.5 |
| Xyl3A or CcXynA | 1.5 |
| ddwater | 5 |
| Total | 10 |

3. Amplification of Target Plasmids

3.1 Preparing LB culture media

• Prepare liquid and solid culture media in separate flasks

| Liquid | | Solid | |
| --- | --- | --- | --- |
| Reagent | Amount | Reagent | Amount |
| ddwater | 294ml | ddwater | 200ml |
| NaCl | 3.3g | NaCl | 2g |
| Yeast | 2g | Yeast | 2g |
| Tryptone | 3.3g | Tryptone | 2g |
| Total | 300ml | Agar | 3g |
|  |  | Total | 200ml |

3.2 Transformation of TOP10 competent cells

• Add 100μl pET28A-xynA, pET28-PKC, pET28a-ccXynA, and pET28a-xyI3A into separate test tubes each containing 100μl
TOP10 competent cells and place on ice for 30 minutes.
• Immerse in 42°C water bath for 60 seconds.
• Place on ice for 2 minutes.
• Add 1ml LB culture media (liquid) to each tube and let sit for 1 hour.
• Inoculate the TOP10 competent cells onto solid LB culture media for 12 hours.
• Pick single colonies and inoculate into 5ml liquid LB culture media that contain 250μg Kanamycin and allow
bacteria to grow for 12 hours.

3.3 Plasmid extraction from TOP10 cells

• Add 250μl Buffer S into an absorption column, then centrifuge at 12000g for 1 minute.
• Centrifuge the 5ml liquid culture medium at 8000g for 2 minutes and discard the resulting liquid (keep the
sediment).
• Add 250μl of SP1 Buffer into the tube and mix well.
• Add 250μl of SP2 Buffer into the liquid and mix gently until it becomes clear.
• Add 350μl SP3 Buffer and mix gently until precipitates form, then centrifuge the tube at 12000g for 10 minutes

• Add resulting liquid to the absorption column, then centrifuge at 8000g for 1 minute and discard the liquid.
• Add 500μl of DW1 Buffer and centrifuge at 9000g for 1 minute and discard the liquid.
• Add 500μl Wash solution and centrifuge at 9000g for 1 minute and discard the liquid. Repeat this step again (total
2 times)
• Centrifuge the absorption column at 9000g for 2 minutes and discard any liquid.
• Add 50μl Elution Buffer and centrifuge at 9000g for 1 minute. Keep the resulting liquid.
• Use the resulting liquid to check for the density of plasmids
• Serialization and Identification of DNA sequences.

4. Protein Expression and Purification

4.1 Preparing Buffers

• Create the following solutions:

| His Buffer A (500ml pH 7.4) | | His Buffer B (250ml pH 7.4) | |
| --- | --- | --- | --- |
| Reagent | Amount | Reagent | Amount |
| Na2HPO4·2H2O | 1.78g | Na2HPO4·2H2O | 0.89g |
| NaCl | 14.6g | NaCl | 7.3g |
| Imidazole | 1.02g | Imidazole | 8.5g |
| ddwater | 500ml | ddwater | 250ml |

4.2 Transformation of Bacteria and Expression of Genes

• Mix 100 ng of pET28A-xynA, pET28-PKC, pET28a-ccXynA, and pET28a-xyI3A with 100μl of E.coli BL21(AI) Strain
competent cells (BL21 competent cells) in separate tubes. Place on ice for 30 minutes.
• Immerse in 42°C water bath for 60 seconds.
• Place on ice for 2 minutes.
• Add 1ml liquid LB culture media and let the culture grow for 12 hours.
• Inoculate the BL21 competent cells onto solid LB culture medium for 12 hours.
• Pick single colonies and inoculate into 5ml liquid LB culture media containing 250μg Kanamycin for 12 hours
• Inoculate into 100ml liquid LB culture media at room temperature for 2-3 hours.
• When the detected OD is at 0.4-0.6, add 0.24mM IPTG and culture overnight.

4.3 Protein purification

• Centrifuge 100ml cultures at 1530g for 15min. Discard the liquid (keep sediment).
• Add 20ml of His Buffer A and mix well so that there is no more sediment at the bottom of the tubes.
• Pack ice in a container and place the tube containing the solution in it, then sonicate the solution for 5 minutes
with settings for 3-second work and 2-second pause; collect the liquid.
• Centrifuge at 9000g or 20 minutes. Keep the liquid but discard of the sediment (keep 50μl samples of both)
• Add 500μl of Ni-NTA beads and let sit for 2 hours at 4˚C.
• Centrifuge at 500g for 5 minutes and collect the sediment (discard the liquid)
• Add 5ml of Buffer A and mix so that the sediment is suspended, then transfer to beads column.
• Add 1ml of His Buffer A 5 times, waiting for all the liquid to filter before each addition.
• Add 500μl of His Buffer B 6 times, waiting for all the liquid to filter before each addition. Collect the final
liquid.
• Use His buffer B to elute protein 500μl (by 5 times) and collect the liquid.
• Measure the concentration of protein in the liquid.

4.4 Verification of Proteins.

4.4.1 SDS PAGE

• Mix protein gel solution (components shown below) and solidify to a
thickness of 1.5mm

|  | Protein gel solution | Buffer | Improved Coagulation Promotion Solution |
| --- | --- | --- | --- |
| Resolving layer | 4ml | 4ml | 80μl |
| Stacking layer | 1ml | 1ml | 20μl |

• Mix 500ml of protein gel buffer

| Reagent | Volume |
| --- | --- |
| Tris-Glycine | 50ml |
| ddwater | 450ml |

• Mix 100ml of coomassie brilliant blue decolorizing solution

| Reagent | Volume |
| --- | --- |
| ethanol | 40ml |
| Acetic acid | 10ml |
| ddwater | 50ml |

• Inject samples and a marker into the wells.
• Add 12.5μl of G250 Loading into target protein and inject into a well
• Electrophoresis at 180V for 1 hour.
• Use coomassie brilliant blue to dye the gel for 1 hour and decolorize for 1 hour.
• Imaging of gel

4.4.2 Enzyme activity measurement by DNS

• Prepare 50ml of MnCl2 solution at a concentration of 5mM
• Add 0.15g Xylan into 30ml of the MnCl2 solution.
• Prepare 1ml each of xynA, PKC, xyl3A, and cCxynA
• Divide the experiment into four groups

|  |  |
| --- | --- |
| Group 1 | 350μl of xylan solution + 50μl of enzyme |
| Group 2 | 70μl of xylan solution + 280μl of MnCl2 solution + 50μl of enzyme |
| Group 3 | 14μl of xylan solution + 336μl of MnCl2 solution + 50μl of enzyme |
| Control group | 350μl of xylan solution + 50μl of enzyme (denature) |

• Immerse all groups in a 37˚C water bath 10 minutes.
• Add 800μl of DNS solution and boil for 5min to denature all enzymes.
• Calculate the enzyme activity.

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Results

1. The pET28a-PKC, pET28a-xynA, pET28a-CcxynA, pET28a-xyl3A (Escherichia coli)

1.1 Construction of the expression plasmids

We design four plasmids: The DNA sequences of PKC, xynA, CcxynA, and xyl3A were inserted into the pET-28a (+)
vector, respectively. In order to build our plasmids, we let the synthetic company synthesize two target gene
fragments, PKC and xynA integrate it into the pUC57 vector. And the target gene fragments CcxynA and xynl3A
integrate them into the pET28a vector.
The certificate of synthesize analysis are as follows:

Certificate of Analysis of pUC57-PKC

Certificate of Analysis of pUC57-xynA

Certificate of Analysis of pET28a-CcxynA

Certificate of Analysis of pET28a-xynl3A

1.1.1 pET28a-PKC

We amplify PKC by PCR, double-enzyme digestion, and inserted into the NheI and HindIII site of pET28a (+) carrier,
respectively, to obtain the plasmids pET28a-PKC. Then the pET28a-PKC transform into DH5α, the colony PCR
identification results show that the construction of pET28a-PKC is successful (Figure 1).

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-results01.jpg)

Figure 1. The PCR identification result of pET28a-PKC
M:2000marker
1.pET28a-xynA (DH5α)
2.pET28a-xynA (DH5α)
3.pET28a-PKC (DH5α)

We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing
comparison results showed that there were no mutations in the ORF region (Figure 2), and the plasmidpET28a-PKC was
successfully constructed.

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-results02.jpg)

Figure 2. The sequencing blast results of the plasmid pET28a-PKC.

The plasmid pET28a-PKC was extracted from DH5α, and transformed it into BL21(DE3). The colony PCR identification
results showed that it was successful (Figure 3).

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-results03.jpg)

Figure 3. The PCR identification result of pET28a-PKC

1.1.2 pET28a-xynA

We amplify xynA by PCR, double-enzyme digestion, and inserted into the XbaI and BamHI site of pET28a (+) carrier, to
obtain the plasmids pET28a-xynA. Then the pET28a-xynA transform into DH5α, the colony PCR identification results
show that the construction of pET28a-xynA is successful (Figure 4).

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-results04.jpg)
Figure 4. The PCR identification result of pET28a-xynA (DH5α).

We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing
comparison results showed that there were no mutations in the ORF region (Figure 5), and the plasmid pET28a-xynA was
successfully constructed.

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-results05.jpg)

Figure 5. The sequencing blast results of the plasmid pET28a-xynA

The plasmid pET28a-xynA was extracted from DH5α, and transformed into BL21(DE3). The PCR identification results
showed that the plasmid pET28a-xynA was successful (Figure 6).

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-results06.jpg)

Figure 6. The PCR identification result of pET28a-xynA BL21(DE3).

1.1.3 pET28a-CcxynA

Because the gene synthesis company delivered a tube of plasmid, we transformed the pET28a-CcxynA into E.coli
BL21(DE3) for expressing proteins. The PCR identification results showed that the transformation of plasmid
pET28a-CcxynA was successful.

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-results07.jpg)

Figure 7. The PCR identification result of pET28a-CcxynA.

1.1.4 pET28a-xyl3A

As a result, we can find that when the length of inserted gene is around 3.5k, we still achieved gene-editing with
casposons. What’s more, as the length of the inserted gene increased, the number of colonies decreased. However,
casposons is still an excellent tool we could use in future research for gene editing.

e) Sanger sequencing to amplify the recombinant plasmids

We transformed the pET28a-xyl3A into E.coli DH5α for amplification. Next, the plasmid pET28a-xynA was extracted from
DH5α, then transformed into BL21(DE3). The PCR identification results showed that the transformation of plasmid
pET28a-xyl3A was successful (Figure 8).

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-results08.jpg)
Figure 8. The PCR identification result of pET28a-xyl3A .

1.2 Protein expression and purification

In order to obtain the four proteins(PKC, xynA, CcxynA, xyl3A), we transferred the recombinant plasmids into E.coli
BL21(DE3), expanded the culture in the LB medium, and added IPTG to induce protein expression when the OD600 reached
0.3-0.5. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to
release the intracellular proteins. Next, we used nickel column purification to purify the protein we wanted.
At this point, we obtained the four proteins solutions we wanted.

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-results09.jpg)

Figure 9. The SDS-PAGE result of the four proteins
M:180KD marker
S：Soluble lysate
P：Precipitation
E：Elution

The molecular weights of xynA, PKC, xyl3A, and CCxynA were 22.87 KD and 48.27 KD, respectively. 84.93KD and 57.0KD;
referring to the marker in Figure 1, we found the four proteins (PKC, xynA, CcxynA, xyl3A) in lane S, indicating
that they were successfully expressed in E. coli BL21 (DE3). The four proteins (PKC, xynA, CcxynA, xyl3A) were also
found in lane P, possibly due to the inactivation of a small number of proteins.

1.3 Test of enzyme activity

Determination of reducing the sugar by DNS method. The absorbance OD540 value of the four purified enzyme solutions
(xynA, PKC, xyl3A, and ccxynA) was measured after color reaction with DNS. The activity of the enzyme can be
converted by the amount of sugar consumed and the working time.
Table 1. Enzyme activity of four solutions (xynA, PKC, xyl3A, and ccxynA)

| Four enzyme solutions | Enzyme activity(U/mL) |
| --- | --- |
| xyl3A | 0.302713345 |
| PKC | 0.267273734 |
| ccxynA | 0.534383396 |
| xynA | 0.139789577 |

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-results10.png)
Figure 10. Enzyme activity of four solutions (xynA, PKC, xyl3A, and ccxynA)

The results showed that the activity of ccxynA enzyme is the highest activity of the four enzymes. The enzyme
activity of xyl3A is higher than that of xynA and PKC. The results indicated that all four proteins were
successfully expressed，and the enzyme activity of four solutions (xynA, PKC, xyl3A, and ccxynA) was active. It
indicated that all four proteins were successfully expressed. According to the different activity values of four
enzymes, we can adopt the strategy of multi-enzyme synergistic degradation to accelerate the degradation rate of
cellulose or xylan in feed, which can be added to feed as a feed additive to improve the ability of animals or
digest and utilize feed, promote animal appetite and improve the quality of animal husbandry products. It can solve
the bottleneck problem of low efficiency and high cost of cellulase hydrolysis.

2. The pSIP403-PUS-xynA (Bifidobacterium) series

2.1 Constructed plasmids

Firstly, we designed the pSIP403-PUS-xynA. We let the xynA to light the plasmid pSIP403-PUS to obtain the
pSIP403-PUS-xynA plasmid.

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-results11.png)

Figure 11. The map of plasmid pSIP403-PUS-xynA.Figure 11. The map of plasmid pSIP403-PUS-xynA.

Next, the xynA and pSIP403-PUS plasmids were digested with HindIII. Figure12 is shown that the plasmid
pSIP403-PUS-xynA was successfully constructed.

![](https://static.igem.wiki/teams/4277/wiki/t-shanghai-united-results12.png)

Figure 12. The results of enzyme digestion identification of pSIP403-PUS-xynA
M：5000bp
1：pSIP403-PUS-xynA (HindIII digestion)
2：pSIP403-PUS-xynA (Undigestion)

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