iGEM 2023 — Team thinker-shenzhen

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/4979/wiki/cover/notebook.png {% endblock %}
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CONTENT

[Experimental preparation for week 1](#a1)
[2 weeks to extract the templates, began to construct the plasmid](#a2)
[3 weeks agarose gel electrophoresis, plastic recycling, double enzyme, recycling](#a3)
[At week 4, competent cells were prepared](#a4)
[At week 5, the cells were connected and transformed](#a5)
[At the 6th week, the two successfully constructed plasmids were extracted and expanded](#a6)
[Validation of alginate lyase AL2 and the effects of temperature and PH on it at the 7th week](#a7)
[Activity of cellulase Bgls, optimal reaction temperature and optimal reaction pH were determined at week 8](#a8)
[Kelp was digested with cellulase Bgls and alginase AL2 at week 9](#a9)
[The effect of SRRz cleavage of the gene was controlled using the arabinose promoter at week 10](#a10)

Experimental preparation for week 1

Experimental plan: Prepare the medium and experimental consumables and activate the plasmid-carrying strain

Experimental procedure:

1. Use of ultra-clean table: open the ultra-clean table, place the centrifuge tube plate, test tube rack, Marker pen, alcohol disinfection and sterilization, and conduct UV irradiation for 30 minutes.

2. Sterilization: Prepare Eppendorf tubes, PCR tubes, and centrifuge tubes, and wrap them with gauze. Fill 2 boxes each of large, medium, small, and gun heads, using Velcro paper to wrap the shell, and using string/rubber band to bind the gun head box. Using pre-prepared LB medium, divide 20 tubes into 50 mL x 2 bottles of LB liquid medium. The above items were sterilized using 121 ° C for 15 min (this process was about 1.5 h). An experimental accident occurred during the filling of liquid medium: the medium was spilled on the ultra-clean table, and it was cleaned up properly afterwards.

3. Activated strains: 1 tube of E. coli DH5α/ empty vector,1 tube of E. coli DH5α/ AL2 gene,1 tube of E. coli DH5α/bgls, 1 tube of DH5α/ arabinose promoter gene, and 1 tube of DH5α/ SRRz cleavage gene. Turn on the ultra-clean table, ventilated, put the sterilized LB test tube into the table, light the alcohol lamp, burn the orifice and plug of the test tube, put 100 μL of preserved bacteria solution into each test tube, add 2.5 μL Amp antibiotic mother solution, burn the orifice and plug of the test tube again. After sealing the tube, the tube was bundled and incubated on a shaker overnight (150 rpm, 37℃).

![](https://static.igem.wiki/teams/4979/wiki/notebook/img1.jpg)

2 weeks to extract the templates, began to construct the plasmid

Experimental plan: Plasmid was extracted and the target fragment was amplified by PCR

Experimental procedure:

1. Extract plasmid: According to the instruction of plasmid extraction kit, four plasmid tubes of Escherichia coli DH5α/ empty vector, one tube of DH5α/ carrying AL2 gene, one tube of Escherichia coli DH5α/bgls, one tube of DH5α/ carrying arabinose promoter gene, and one tube of DH5α/ carrying SRRz cleavage gene were extracted and centrifuged for 1min, then the precipitate was collected. p1 was added and resuspended, p2 was mixed upside down, p3 was mixed upside down, and then centrifuged for 10min. 500μL BL reagent was added to the adsorption column, the waste solution was poured, the supernatant after centrifugation as described above was added, and the mixture was rinsed twice using pw rinse solution, and centrifuged for 1min each time. EB solution 50μL was added to the adsorption column membrane and centrifuged for 2min. The recovered DNA concentration was determined using Nanodrop and the DNA was stored at -20 ° C. In this step, most people successfully extracted the plasmid.

![](https://static.igem.wiki/teams/4979/wiki/notebook/img2.jpg)

2. Dilute the primers

The dry powder primer was centrifuged at 12,000 RPM for 1min, and the corresponding amount of distilled water was added to dilute the primer to the appropriate concentration

![](https://static.igem.wiki/teams/4979/wiki/notebook/img3.jpg)

3.PCR reaction:

(1) The empty vector of target gene, AL2 gene,bgls, arabinose promoter gene were amplified by high fidelity enzyme Pfu, and the DNA fragment of the gene was cleaved by SRRz. The reaction system is as follows:

Pfu 0.5 μL

5 μL of 10xPfu buffer

Primer 1 1 μL

Primer 2 1 μL

Template (plasmid extracted above) : 1μL

25μL dNTP Mixture

20 μL of sterilized water

(2) Perform the PCR amplification procedure

At 98 ° C for 5 min

The following 3-step reaction was performed for 30 cycles

98℃ 10s

55℃ 5s

72℃ 1min/kb

72℃ 8 min

The plasmids, primers and products required for PCR are shown in the table below:

3 weeks agarose gel electrophoresis, plastic recycling, double enzyme, recycling

Experimental plan: Agarose gel electrophoresis, gel recovery, double enzyme digestion, recovery

Experimental procedure:

1 Agarose gel electrophoresis: The above PCR products were added to a previously prepared agarose gel with 5μL DL 5000 DNA marker and subjected to electrophoresis (120V, 20 min). At the end of electrophoresis, the gel imaging system was used for observation. The target DNA gel was cut, placed in a 2 mL centrifuge tube, and labeled.

2. Gel recovery: The PCR product DNA gel was recovered according to the instructions of the gel recovery kit

Add 500 microliters of equilibrium solution BL to the adsorption column CA2 and centrifuge for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube. A single band of target DNA was cut from the agarose gel into a clean centrifuge tube. Add an equal volume of solution PN to the gel block and place in a water bath at 50 ° C until the gel block is completely dissolved. The solution obtained in the previous step was added to another adsorption column CA2, placed at room temperature for 2 min, and centrifuged for 1min. Add 600 microliters of bleach solution PW to the adsorption column CA2 (please check whether absolute ethanol has been added before use), centrifuge for 1min, repeat rinsing twice, and let dry. Add an appropriate amount of elution buffer EB to the middle position of the adsorption membrane and place it at room temperature for 2 min. The DNA solution was collected by centrifugation for 2min. The recovered DNA concentration was determined using Nanodrop.

3 Double digestion: EcoRI and XhoI were used to double digest the extracted plasmid pET23b and the amplified target DNA fragment. 5x20 μL reaction. The 20 μL digestion system is as follows:

1 μg of DNA

1 μL of EcoRI

1 μL XhoI

4 μL 10xY buffer

H2O up to 20μL

The enzyme was digested at 37 ° C for 4h

4 Agarose gel electrophoresis: The digested product was added to the agarose gel, 5μL of DL 5000 DNA marker was added, and electrophoresis was performed (120V, 20 min). At the end of electrophoresis, the gel imaging system was used for observation. The target DNA gel was cut, placed in a 2 mL centrifuge tube, and labeled. The product was extracted successfully, but the amount was small. After that, the second gum recovery was carried out.

![](https://static.igem.wiki/teams/4979/wiki/notebook/img4.jpg)

At week 4, competent cells were prepared

Experimental plan: E. coli DH5α and Rosetta were activated to prepare competent cells

Experimental procedure:

1. Activated strains: including E. coli DH5α and Rosetta. Turn on the ultra-clean table, ventilate, put the sterilized LB test tube into the table, light the alcohol lamp, burn the test tube mouth and test tube stopper, connect 100 μL of preserved bacteria solution into each test tube, and burn the test tube mouth and test tube stopper again. The test tube was closed, bundled, and incubated on a shaker overnight (220 rpm, 37℃).

Experimental procedure:

2. Preparation of competent cells:

(1) Open the ultra-clean stage, put in 1 mL tip, 200 μL tip, 5 mL tip, 2mL and 30 mL centrifuge tubes, and 2 x 50 mL LB medium for UV irradiation. The 0.1 M cacl 2 and 15% 0.1 M cacl 2 solution were pre-cooled in a refrigerator at 4 ° C.

(2) DH5α and Rosetta competent cells were prepared as follows: pre-activated and repacked DH5α and Rosetta bacteria were precooled in a centrifuge at 4 ° C, centrifuged at 5000 g for 10 min, the supernatant was discard, and 800 microliters of 0.1 M CaCl2 solution was added to an ice bath for 40 min. After centrifugation again (4000 g, 10 min), the supernatant was discarded, and 100 microliters of 15% glycerol 0.1 M CaCl2 were added to each tube to resuspend the bacteria. Aliquots of competent cells into 2 mL centrifuge tubes of 100μL per tube were dispensed on ice. After labeling, the competent cells were stored in the refrigerator at -80℃.

At week 5, the cells were connected and transformed

Experimental plan: Prepare solid AGAR plates containing antibiotics, perform ligation reactions, and transform ligation products

Experimental procedure:

1. Preparation of antibiotic plates: LB solid medium (4.5 g AGAR added to 300 mL LB liquid medium) was prepared and sterilized at 121 ° C for 15 min by autoclave. After cooling to about 60℃, add 150 μL of 100mg/mL Amp antibiotic, shake evenly, and pour into the disposable Petri dish (about 10-15 plates).

2. Ligation reaction: double enzyme digestion of the vector and the target fragment was connected. The ligation system is as follows (Note: This system is an empirical system, and the optimal linking vector/target fragment volume ratio can also be calculated according to the instructions) :

T4 DNA ligase 1 μL

2 μL of 10 x ligation buffer

1 μL of Linear plasmid

Insert 6 μL of DNA

Connect for 1h at 16 ° C using a PCR apparatus. pET23b-insert was obtained, and the ligation product was stored at -20 ° C.

3. Transformation of ligation products: 10 μL of the above ligation products were added to 100 μL competent cells, followed by an ice bath for 30 min, a water bath at 42 ° C for 60 s, and an ice bath for 5 min. Then, 1 mL LB liquid medium was added and incubated for 1 h for recovery (37 ° C, 150 rpm). In an ultra-clean stage, 100 μL of recovery solution was taken and the Amp antibiotic plate was coated with a sterile coating rod.

![](https://static.igem.wiki/teams/4979/wiki/notebook/img5.jpg)

At the 6th week, the two successfully constructed plasmids were extracted and expanded

Experimental plan: colony PCR verification, expansion of positive clones, extraction and verification of the correct plasmid and transformation

Experimental procedure:

1.PCR verification: Six colonies were labeled on each plate. Half of the colonies were picked and used as templates for colony PCR using Taq enzyme. The primers are 1,2. Reaction systems and procedures were carried out according to the instructions. Configure a 50 mL agarose gel, using DL 2000 DNA marker, and perform electrophoresis. The correct size of the band indicates that the plasmid was successfully constructed

2. Expanded culture: The correct colonies were selected and added to 5 mL LB liquid medium, and 2.5 μL Amp antibiotic was added to expand the culture.

3. Plasmid extraction: Two kinds of plasmids were extracted according to the instruction of plasmid extraction kit. The bacterial solution was centrifuged for 1min and then the precipitate was taken. p1 was added separately and resuspended, p2 was mixed upside down, p3 was mixed upside down, and centrifuged for 10min. 500μL BL reagent was added to the adsorption column, the waste solution was poured, the supernatant after centrifugation as described above was added, and the mixture was rinsed twice using pw rinse solution, and centrifuged for 1min each time. EB solution 50μL was added to the adsorption column membrane and centrifuged for 2min. The recovered DNA concentration was determined using Nanodrop and the DNA was stored at -20 ° C. In this step, most people successfully extracted the plasmid.

4. Transformation: Verify the correct plasmid 1 transformation into expression Escherichia coli.

Validation of alginate lyase AL2 and the effects of temperature and PH on it at the 7th week

Experimental procedure:

1. Verification of alginate lyase AL2:

The expanded bacterial solution was centrifuged to collect cell precipitates and resuspended in Tris-HCl (pH 7.4). Cell lysates were then obtained by lysis by ultrasonic fragmentation (150W, 1s sonication, 3s interval, 20 min in total). To verify the function of alginate lyase, 0.9 mL of substrate solution (0.5% (w/v) of alginate (Solarbio), 50mM Tris-HCI buffer, 200mM NaCl, pH 7.3) was mixed with 0.1 mL of cell lysate. The cells were incubated at 37°C for 30 min before sampling. The 3, 5-dinitrosalicylic acid (DNS) method was used to determine the concentration of reducing sugar. Absorbance at 540nm was recorded to calculate activity. One enzyme activity unit (U) was defined as the amount of enzyme required to release 1μmol of reducing sugar per minute. Protein concentration was determined using Bradford (using fetal bovine serum protein as a standard), and the specific activity of alginate lyase was defined as U/mg. All experiments were performed in triplicate and data will be expressed as mean plus SD.

Controls were experiments performed using untransformed E. coli Rosetta. The activity of the control group was 5 U/mg, which may be due to the weak effect of other cell components on the brown algae gum or the background noise of the experiment. Under our experimental conditions, the specific activity of the crude enzyme solution of alginate lyase was 323 U/mg. This means that each milligram of the crude enzyme solution is capable of releasing 323 micromoles of reducing sugar per minute. This result demonstrated the efficient activity of alginate lyase in our engineered bacteria and provided strong support for further application.

![](https://static.igem.wiki/teams/4979/wiki/notebook/img6.jpg)

2. Effect of temperature on alginate lyase:

To determine the optimal reaction temperature for alginate lyase, cell lysates were diluted using Tris-HCl buffer pH 7.4 and mixed with 0.5% (w/v) alginate solution. After incubation at 25 ° C, 37 ° C, and 45 ° C for 30 min, the concentration of reducing sugar was determined using the 3, 5-dinitrosalicylic acid (DNS) method.

![](https://static.igem.wiki/teams/4979/wiki/notebook/img7.jpg)

3. Effect of PH on alginate lyase:

To determine the optimal reaction pH for alginate lyase, we mixed cell lysates with 0.5% (w/v) alginate solution using citrate buffer (10 mM) at pH 6.1 and Tris-HCl buffer (10 mM) at pH7.4, 8.5, and 10.2. After incubation at 37 ° C for 30 min, the concentration of reducing sugar was determined using the 3, 5-dinitrosalicylic acid (DNS) method. Buffer pair groups were set and no enzyme was added, only substrate and buffer were added. This measures the background absorbance of each buffer and subtract this background value from the experimental group's data.

Note: When Tris is reacted with HCl, Tris-hcl can be obtained. The pH of the Tris-HCl buffer ranges from 7.0 to 11. The citrate buffer uses a reaction of sodium citrate and citric acid ranging from 3 to 6.2.

![](https://static.igem.wiki/teams/4979/wiki/notebook/img8.jpg)

Activity of cellulase Bgls, optimal reaction temperature and optimal reaction pH were determined at week 8

Experimental procedure:

1. Determination of cellulase Bgls activity:

The engineered strains were grown in LB medium at 37°C for three days followed by centrifugation at 13,000 rpm for 5 min. The bacterial precipitate was collected, sonicated and resuspended in PBS(10 mM, pH7.4), and 1 mL of the supernatant was mixed with 1 mL of 1% carboxymethyl cellulose (CMC, dissolved in PBS) and incubated at 37°C for 30 min with shaking (120 rpm). 1 mL of DNS reagent was added and the mixture was boiled for 5 min before absorbance was measured at 540 nm. A unit of enzyme activity was defined as per µmol of reducing sugar produced per minute. Total protein concentration was determined according to the Bradford method. A standard curve was drawn based on the different concentrations of bovine serum albumin (BSA) performed. The specific activity of cellulase was calculated by expressing the final product concentration (μmol reducing sugar/min) as units divided by the total protein (mg) of the sample. The control group was the experiment performed using untransformed E. coli Rosetta. The activity of the control group was 0.5 U/mg, which may have been the background noise of the experiment. Under our experimental conditions (37 ° C, pH 7.4), the specific activity of the crude enzyme solution of Bgls was 11.45 U/mg. This means that each milligram of the crude enzyme solution is capable of releasing 12 micromoles of reducing sugar per minute. This result confirmed the cellulase activity in our engineered strain and provided strong support for the co-application of cellulase and alginase.

![](https://static.igem.wiki/teams/4979/wiki/notebook/img9.jpg)

2. Optimal reaction temperature of cellulase Bgls

To determine the optimal reaction temperature for cellulase, we mixed the crude enzyme solution and 1% CMC solution and determined the reducing sugar concentration using the 3, 5-dinitrosalicylic acid (DNS) method after incubation at 25 ° C, 37 ° C, and 55 ° C for 30 min.

![](https://static.igem.wiki/teams/4979/wiki/notebook/img10.jpg)

3. Optimal reaction pH of cellulase Bgls

To determine the optimal reaction temperature for cellulase, we mixed the crude enzyme solution and 1% CMC solution at 37 ° C. After incubation at pH 5.8, 6.5, and 7.4 (PBS buffer) for 30 min, the concentration of reducing sugar was determined using the 3, 5-dinitrosalicylic acid (DNS) method. The optimum pH of Bgls was 6.5.

![](https://static.igem.wiki/teams/4979/wiki/notebook/img11.jpg)

Kelp was digested with cellulase Bgls and alginase AL2 at week 9

Experimental procedure:

Wash the kelp thoroughly using purified water to remove any accumulated dirt and dry it at 65°C for 2 hours. Soak 1g of dry kelp in water for 2 hours and cut into 1cm2 pieces. Crude enzyme solutions of cellulase and alginase were prepared using Tris-HCI (pH 7.4). 1g of dried kelp was mixed with 20 mL of the reaction system (10 mM Tris-HCI buffer, 10 mL of alginase solution, and different volumes of cellulase solution). After 24 hours of incubation at 37°C at 180 rpm. Samples were taken, kelp was filtered and washed, and then dried at 65°C to constant weight. The degradation rate was calculated by comparing the kelp weight before and after the treatment. All experiments were performed in triplicate and data will be expressed as mean plus SD.

![](https://static.igem.wiki/teams/4979/wiki/notebook/img12.jpg)

The effect of SRRz cleavage of the gene was controlled using the arabinose promoter at week 10

Experimental procedure:

The recombinant plasmid was transformed into E. coli Rosetta competent cells. Positive transformants were screened on LB AGAR plates supplemented with ampicillin. Individual colonies were inoculated into LB broth and cultured with various concentrations of arabinose to induce SRRz expression. Bacterial growth was monitored until 28 h to assess the inhibitory effect of SRRz expression. All experiments were repeated three times, and data are presented as mean ± standard deviation (SD).

![](https://static.igem.wiki/teams/4979/wiki/notebook/img13.jpg)

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/4979/wiki/cover/engineering.png {% endblock %}
{% block page\_content %}

CONTENT

[Overview](#a1)
[Cycle 1: alginate lyase](#a2)
[Design](#b1)
[Build](#b2)
[Test](#b3)
[Learn](#b4)
[Cycle 2: Addition of Bacterial Cellulase to increase efficiency](#a3)
[Design](#b5)
[Build](#b6)
[Test](#b7)
[1.Validation of Cellulase Bgls](#c1)
[2.The optimal reaction temperature for Cellulase Bgls](#c2)
[3.The Optimal pH Value of reaction for Cellulase Bgls](#c3)
[4.Digest kelp by cellulase Bgls and alginase AL2.](#c4)
[Learn](#b8)
[Cycle 3: Utilizing SRRz cleavage gene as biosafety measure and barrier breaker](#a4)
[Design](#b9)
[Build](#b10)
[Test](#b11)
[Cycle 4: Exploring the Potential of Engineered Bacterial Fermentation Products
from Kelp as Seed Germination Enhancers](#a5)
[Design](#b12)
[Build](#b13)
[Test](#b14)
[Learn](#b15)
[Conclusion](#a6)

Overview

Alginate is a high molecular weight polysaccharide extracted from seaweed, widely used in various fields such
as food, medicine, cosmetics, and agriculture. However, due to its non-degradable nature, its production and use
lead to significant waste and environmental pollution. Therefore, finding an efficient method for alginate
degradation is crucial. Kelp is a typical brown algae rich in alginate and cellulose polysaccharides. We
employed microbial alginate lyase and cellulase enzymes to hydrolyze kelp, aiming to enhance its degradation
efficiency and product quality. The microbial alginate lyase can break down alginate into smaller
oligosaccharides and monosaccharides, while cellulase can decompose cellulose into smaller cellulose molecules.
Additionally, cellulase helps increase the solubility of hydrolysis products. We overexpressed alginate lyase
(AL2) and bacterial cellulase bgls separately and verified their functions. To enhance the safety of engineered
bacteria and release alginate lyase and bacterial cellulase, we also studied the lysis effect of SRRz under the
control of the arabinose promoter. Moreover, the oligosaccharides produced by alginate lyase can promote plant
growth and enhance their stress resistance, while the sugars produced by cellulase can provide energy for
plants. To verify the benefits of the fermentation products from the engineered bacteria on plants, we applied
these recombinant engineered bacteria in kelp fermentation, treated seeds with the fermentation liquid, and then
tested their germination rate.

Cycle 1: alginate lyase

Design

In order to maximize the utility of alginate oligosaccharides, we first need to produce them. Alginate
oligosaccharides are formed through the hydrolysis of alginate. After reviewing the literature, we discovered
that alginate lyase can perfectly accomplish this task. As a biological enzyme, it offers advantages such as
mild reaction conditions, ease of operation, low energy consumption, high yield, high specificity, and
environmental friendliness. After a thorough comparison, we found that enzymatic degradation has significant
advantages over chemical and physical degradation methods. Therefore, we have decided to use it as our method
for hydrolyzing alginate.

![](https://static.igem.wiki/teams/4979/wiki/engi/img1.jpg)

Figure 1. The mechanism of how alginate lyase hydrolyzes alginate.

Build

![](https://static.igem.wiki/teams/4979/wiki/engi/img2.jpg)

Figure 2. The gene circuit for AL2 overexpression.

Test

![](https://static.igem.wiki/teams/4979/wiki/engi/img3.jpg)

Figure 3. Gel electrophoresis of AL2 .

![](https://static.igem.wiki/teams/4979/wiki/engi/img4.jpg)

Figure 4 Experimental Results on AL2 .

![](https://static.igem.wiki/teams/4979/wiki/engi/img5.jpg)

Figure 5. The mechanism of how alginate lyase hydrolyzes alginate.

The recombinant vector was transformed into E. coli Rosetta cells ，the cell pellet was collected by
centrifugation and resuspended in Tris-HCl (pH 7.4). The cells were then lysed through ultrasonication, yielding
the cell lysate. The total protein concentration in the lysate was determined using the Bradford assay kit . To
validate the function of the alginate lyase, 0.9 mL of substrate solution alginate , 50 mM Tris-HCI buffer, 200
mM NaCl, was mixed with 0.1 mL of cell lysate and incubated at 37°C for 30 minutes before sampling. The
concentration of reducing sugars was determined using the 3,5-dinitrosalicylic acid (DNS) method. The absorbance
at 540nm was recorded to calculate the activity. The activity in the control group was measured at 5 U/mg. Under
our experimental conditions, the specific activity of the alginate lyase crude extract was determined to be 323
U/mg in figure4A. This means that each milligram of crude extract can release 323 micromoles of reducing sugar
per minute. This result demonstrates the high efficiency of the alginate lyase in our engineered bacteria,
providing strong support for further applications.

To determine the optimal reaction temperature for alginate lyase, we diluted the cell lysate with Tris-HCl
buffer at pH 7.4 and mixed it with a 0.5% (w/v) solution of alginate. After incubating at 25°C, 37°C, and 45°C
for 30 minutes, we measured the concentration of reducing sugar using the 3,5-dinitrosalicylic acid (DNS)
method. To determine the optimal reaction pH for alginate lyase, we mixed the cell lysate with a 0.5% (w/v)
solution of alginate using different buffer solutions. We used a 10 mM citrate buffer at pH 6.1, as well as
Tris-HCl buffers at pH 7.4, 8.5, and 10.2. After incubating at 37°C for 30 minutes, we measured the
concentration of reducing sugar using the 3,5-dinitrosalicylic acid (DNS) method.The result is shown in Figure
4B and 4C. The optimal reaction temperature for AL2 is 37°C, and the optimal pH condition is 7.4.

Learn

The results have shown that alginate lyase is capable of hydrolyzing alginate efficiently and in a sustainable
way. However, since algae in seaweed, not only do they have large amounts of alginate, but they also contain
tons of cellulose that might surround the alginate inside the cells of algae and seaweed, preventing the
alginate lyase from hydrolyzing as much alginate as possible. Therefore, we want to see if there is a way to
hydrolyze the cellulose effectively.

Cycle 2: Addition of Bacterial Cellulase to increase efficiency

Design

After brainstorming and literature collection within the group, we found that bacterial cellulase is very
suitable for the task of degrading cellulose. Bacterial Cellulase, an enzyme produced by bacteria, can break
down cellulose into a usable carbon source. Bacterial Cellulase belongs to a complex enzyme system, including
Endoglucanase (EC 3.2.1.4), Exoglucanase (EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21), all of which play roles
in cellulose degradation. With the three enzymes working together, the end product would be glucose.

![](https://static.igem.wiki/teams/4979/wiki/engi/img6.jpg)

Figure 6. Mechanism of Cellulase.

Build

![](https://static.igem.wiki/teams/4979/wiki/engi/img7.jpg)

Figure 7. The gene cirtuit design of bgls overexpression system.

![](https://static.igem.wiki/teams/4979/wiki/engi/img8.jpg)

Figure 8. Gel electrophoresis of bgls.

Test

1.Validation of Cellulase Bgls

2.The optimal reaction temperature for Cellulase Bgls

3.The Optimal pH Value of reaction for Cellulase Bgls

4.Digest kelp by cellulase Bgls and alginase AL2.

![](https://static.igem.wiki/teams/4979/wiki/engi/img9.jpg)

Figure 9. Experimental Results on bgls .

To evaluate the expression and activity of Bacillus subtilis cellulase Bgls in E. coli Rosetta, we first
synthesized the bgls gene and cloned it into the pET23b vector. Engineered E. coli Rosetta was cultured in LB
broth medium for 3 days at 37°C, and then centrifuged at 13,000 rpm for 5 minutes. Cell pellet was resuspended
in PBS buffer (10 mM, pH 7.4) and then lysed through ultrasonication (150 W, 1s sonication with 3s intervals,
for a total of 20 minutes). 1 ml of the supernatant (crude enzyme solution) was mixed with one ml of 1%
carboxymethylcellulose (CMC, Sigma-Aldrich) solubilized in PBS buffer (10 mM, pH 7.4) and incubated at 37°C for
30 minutes under shaking (120 rpm). One ml of 3,5-dinitrosalicylic acid (DNS) reagent was added and the mixture
was boiled for 5 minutes, then the absorbance was measured at 540 nm.

Under our experimental conditions, the specific activity of bgls was determined to be 11.45 U/mg in figure8A.
Translation: To determine the optimal reaction temperature of cellulase, we mixed crude enzyme solution with a
1% CMC solution and incubated it at 25°C, 37°C, and 55°C for 30 minutes. After that, we used the
3,5-dinitrosalicylic acid (DNS) method to measure the concentration of reducing sugars. We found that the
optimal reaction temperature for cellulase was 37°C, as shown in Figure 9B.

Furthermore, to determine the optimal reaction pH of cellulase, we mixed the crude enzyme solution with a 1%
CMC solution under 37°C conditions. After incubation for 30 minutes at pH 5.8, 6.5, and 7.4 (in PBS buffer), we
measured the concentration of reducing sugars using the DNS method. The optimal pH for Bgls was found to be 6.5,
as shown in Figure 9C.

![](https://static.igem.wiki/teams/4979/wiki/engi/img10.jpg)

Figure 10. Digestion results of kelp using cellulase Bgls and alginase AL2.

To verify the digestion efficiency of AL2 and bgls, the kelp was thoroughly washed with purified water to
remove any accumulated dirt and dried at 65°C for 2 hours. 1g of dried kelp was soaked in water for 2 hours and
then cut into 1cm2 pieces. The crude enzyme solution of cellulase and brown algae enzyme was prepared using
Tris-HCI (pH 7.4). 1g of dried kelp and 20 mL reaction mixture containing 10 mM Tris-HCI buffer, 10 mL brown
algae enzyme solution, and varying volumes of cellulase solution were prepared. After incubation at 37°C and 180
rpm for 24 hours, samples were taken. The kelp was filtered, washed, and dried at 65°C until a constant weight
was achieved. Degradation rates were calculated by comparing the weight of the kelp before and after treatment.
As shown in Figure 10, we found that the degradation rate of the kelp increased progressively with increasing
volume of the crude cellulase solution. When the volume of crude cellulase solution reached 10 mL, the
degradation rate of the kelp reached 59.11%.

Learn

By the results given above, we have proven that bacterial cellulase is capable of breaking down cellulose and
forming glucose in a sustainable and highly efficient way. However, as the bacterial cellulase and alginate
lyase are expressed through genetic engineering in Escherichia coli and our substrates are located outside the
bacterial cells, we need to lyse the bacteria to allow the expressed enzymes to come into contact with the
substrates and facilitate the reaction. Additionally, as we are incorporating Escherichia coli in our fertilizer
production process, ensuring biosafety is of utmost importance. Therefore, we would need to find a way to make
sure all E. coli die before the alginate oligosaccharide (as an end product) gets used as a fertilizer.

Cycle 3: Utilizing SRRz cleavage gene as biosafety measure and
barrier breaker

Design

The recombined E.coli left will cause contaminations to the environment, we decided to insert SRRz lytic gene,
which are linked genes consist of perforin gene S, phage lysosome(transglycosidase) gene R, and gene RZ, into
the plasmid to control bacteria to decompose by itself.

Build

We used linked genes composed of the perforin gene S, the phage lysozyme (transglycosidase) gene R, and the
gene RZ. Arabinose promoter, BBa B0015 terminator is used. The constructed plasmid was introduced into
Escherichia coli Rosetta receptor cells.

![](https://static.igem.wiki/teams/4979/wiki/engi/img11.jpg)

Figure 11. Gene circuit design of araBAD promoter and SRRz cleavage gene.

Test

![](https://static.igem.wiki/teams/4979/wiki/engi/img12.jpg)

Figure 12. Gel electrophoresis of araBAD promoter and SRRz.

![](https://static.igem.wiki/teams/4979/wiki/engi/img13.jpg)

Figure 13. Experimental Results on arabinose promoter and SRRz cleavage gene.

The figure 13A shows the growth of E.coli Rosetta in 28 hours with different concentration of arabinose. It is
obvious that the concentration of arabinose is inversely proportional to the absorption value, in which when
there is 0 mM arabinose, the absorption value at OD600 is about 2.25, but when there is 1 mM arabinose, the
absorption value is about 0.2. The results show the significant decrease of the amount of bacteria as arbinose
concentration increases, which proves the obvious effect of arabinose and SRRz genes.

The figure 13B shows the rate at which the bacteria proliferate under different concentrations of arabinose.
The horizontal coordinate indicates different concentrations of arabinose, and the vertical coordinate indicates
the absorption value of 600nm wavelength detected by the bacteria over a 28-hour period. The higher the
absorption value, the more quantity. The results showed that with the increase of arabinose concentration, the
light absorption value decreased and the number of bacteria decreased. We use One-way ANOVA to determine the
statistical significance between induced and non-induced culture at the 28th hour, and p < 0.05 was considered
statistically significant. The results showed that bacterial growth inhibition was enhanced with the increase
of induction intensity.

The application of the SRRz cleavage gene in the field of biotechnology has garnered widespread attention.
Especially in terms of the biosafety of engineered bacteria, this gene has demonstrated significant
potential. When engineered bacteria are exposed to an environment with 1 mM of arabinose, the expression of
SRRz is activated, leading to the complete lysis of the bacteria. This means that at this concentration of
arabinose, engineered bacteria cannot survive, ensuring they do not inadvertently leak into the external
environment, posing a potential threat to the ecosystem. However, under conditions of 0.1 mM arabinose, the
expression level of SRRz is not sufficient to fully inhibit bacterial growth. This offers us a unique
opportunity where, under these conditions, bacteria can release alginate lyase and cellulase. These enzymes
are crucial for the degradation of kelp, as they can break down the complex polysaccharides in kelp,
providing valuable raw materials for industry and agriculture. In summary, by precisely controlling the
concentration of arabinose, we can utilize the SRRz cleavage gene to achieve biosafety and barrier
disruption in engineered bacteria, offering a new strategy for biotechnological applications.

![](https://static.igem.wiki/teams/4979/wiki/engi/img14.jpg)

Figure 14. Evaluating the performance of engineered strains in kelp fermentation.

To evaluate the effectiveness of engineered strains in kelp fermentation, we will apply the engineered strains
to actual fermentation in the future. We added the SRRz lysozyme gene to the aforementioned two engineered
strains, controlled by an Arabidopsis sugar promoter. To assess the performance of strains Bgls/SRRz and
AL2/SRRz in kelp fermentation, we set up two groups. The CK group was not inoculated with any strains, while the
Bgls/SRRz and AL2/SRRz group was co-inoculated with strains Bgls/SRRz and AL2/SRRz. The kelp was thoroughly
cleaned with purified water to remove any accumulated dirt and dried at 65°C for 2 hours. Then, 1,000 g of dried
kelp was soaked in water for 2 hours, chopped, and added to a 20 L fermentation bottle. Subsequently, 5 L of LB
medium (with 100 μg/mL ampicillin) and 10% (v/v) of the engineered strains (OD600 = 0.6) were added. Based on
the in vitro testing results of the SRRz lysozyme strain, 0.1 mM of L-arabinose was added to induce bacterial
lysis and allow the engineered strains to continue growing. Fermentation was carried out at 37°C and 150 rpm.
Tissue culture membrane sealing was used to ensure oxygen supply. Every 48 hours, 50% of fresh LB medium
(supplemented with 100 μg/mL ampicillin) was replaced in a laminar flow hood. After two weeks, the kelp in the
fermentation bottle was filtered, washed, and dried at 65°C to a constant weight. The degradation rate was
calculated by comparing the weight of the kelp before and after treatment. All experiments were conducted in
triplicate, and the data will be expressed as mean±SD. It can be observed that the degradation rate of 1 kg of
kelp by the mixed engineered strains Bgls/SRRz and AL2/SRRz was approximately 60% in the second week, as shown
in Figure 14.

Cycle 4: Exploring the Potential of Engineered Bacterial Fermentation
Products from Kelp as Seed Germination Enhancers

Design

We designed an experiment to explore the potential of fermentation products from engineered bacteria on kelp as
fertilizers. Several types of seeds were chosen as test subjects to evaluate the impact of the fermentation
products on their germination rates.

Build

To obtain the fermentation products, we fermented kelp using engineered bacteria. After fermentation, we
filtered the products using a 0.22-micron membrane to remove any potential bacteria and then diluted it to a
concentration of 10%.

Test

Seeds were divided into two groups: the test group seeds were soaked in the filtered fermentation product,
while the control group seeds were soaked in clean water. After a 24-hour soak, they were sown in cultivation
trays. On the third day, we observed and recorded the germination status of each seed group. Notably, pine
willow seeds treated with fermentation products showed a germination boost from 74% to 90%. Similarly, soybean
seeds exhibited an increase from 76% to 84%. However, wheat, barley and triticale seeds did not perform well in
our tests, with low germination rates in both the control and treatment groups. This could be attributed to our
cultivation methods or conditions.

Table 1. Germination rate of different treatment groups

![](https://static.igem.wiki/teams/4979/wiki/engi/img15.jpg)

Learn

The fermentation products of engineered bacteria indeed promote germination in certain seeds, providing
preliminary evidence for their potential application in agriculture. Additionally, experimental conditions, such
as the concentration of fermentation products and soaking duration, may significantly influence the results,
suggesting that we need to be more meticulous in selecting and optimizing these conditions in future design and
build phases. Given the low germination rate of some seeds, a more comprehensive cultivation condition should be
considered in future testing stages. Lastly, we need to further investigate the long-term effects of
fermentation products when applied in agriculture.

Conclusion

In conclusion, the experiment data proves the signinficant effects of combining alginate lyase gene and
bacterial cellulase together to decompose alginate polysaccharide to oligosaccharide, as well as the SRRz
suicide gene, which enables engineered bacteria to be killed to prevent biological contamination. In order of
the environmentally friendly and sustainable advantages, our system will be appropriate to be applied in
fertilization fields, as the alginate oligosaccharides benefit the plants.

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