iGEM 2024 — Team qs-fmi

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# Note

### Content

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[Experiment 1](#section1)
[Experiment 2](#section2)
[Experiment 3](#section3)
[Experiment 4](#section4)
[Experiment 5](#section5)

### Experimental Notes on Laccase Production by Recombinant BL21 Escherichia coli for Degrading Estradiol

##### Experiment 1: Cultivation of Recombinant BL21 Escherichia coli

Experimental Dates: 2024/9/12 - 2024/9/14

Experimental Purpose:To achieve the optimal growth and expression state of the target microorganism through precise cultivation conditions, so as to obtain high-quality cultured products and provide reliable experimental materials for subsequent research and applications.

1. Experimental Steps and Processes

In the initial stage, under a strict aseptic operation environment, a single colony on the medium was precisely picked into the liquid LB medium (containing 100μg/mL ampicillin) using a sterile inoculation tool. Subsequently, the culture system was placed in a constant temperature incubator with the temperature precisely controlled at 37℃ and oscillated at a stable speed of 150 rpm for overnight cultivation. The next day, the solution was filtered through a 0.45μm filter membrane to ensure sterility, and then IPTG with a final concentration of 0.5mM and Cu2+ with a final concentration of 0.5 mM were carefully added. The temperature of the incubator was adjusted to 30℃, and the induction cultivation continued for 16 hours.

2. Observation and Recording

During the overnight cultivation at 37℃, the growth signs of bacteria were monitored regularly by visual inspection and microscopic examination. After adding the inducer, the changes in various parameters of the culture, including color, turbidity, and colony morphology, were closely observed. Based on relevant references, it is expected that the optimal cultivation effect can be achieved during the following 16-hour induction cultivation.

3. Experimental Analysis

Each step of this experiment has been carefully designed and strictly controlled to ensure the reliability and reproducibility of the experimental results. During the process of picking a single colony, a high degree of accuracy and aseptic operation awareness are required to avoid contamination and mixing with other microorganisms. The selection of appropriate cultivation temperature and rotation speed is conducive to the rapid growth and metabolism of microorganisms. The timing and concentration of adding inducers are also crucial as they will directly affect the expression level and quality of the target protein.

4. Experimental Summary

Microbial cultivation experiments require a high level of professional knowledge and skills, as well as a rigorous experimental attitude and operational norms. Every detail may have a significant impact on the experimental results, so it is necessary to be vigilant and focused at all times. At the same time, continuous thinking and analysis during the experiment are required, and the experimental protocol should be adjusted in a timely manner to deal with various possible problems.

##### Experiment 2: Detection of Laccase Activity in Recombinant BL21 Escherichia coli

Experimental Dates: 2024/9/14 - 2024/9/17

Experimental Purpose：To determine the laccase activity.

1. Experimental Principle

Laccase can decompose the substrate ABTS to produce ABTS radicals, and the absorbance coefficient of ABTS radicals at 420 nm is much larger than that of the substrate ABTS. The laccase activity can be calculated by measuring the increasing rate of ABTS radicals.

2. Experimental Steps

2.1 Sample Processing

The operation was carried out according to the ratio of the number of bacteria (108 per mL) to the volume of the extraction liquid (mL) being 20:1. Under ice-bath conditions, the cells were disrupted using an ultrasonic device with a power of 300 W, with an ultrasonic time of 3 seconds, an interval of 7 seconds, and a total time of 3 minutes. Subsequently, the cells were centrifuged at 4℃ with a centrifugal force of 10,000 g for 10 minutes.The supernatant was placed on ice for testing, and the culture medium was directly tested.

2.2 Detection Steps

The spectrophotometer was preheated for more than 30 minutes in advance, the wavelength was adjusted to 420 nm, and zeroed with distilled water.The water bath was adjusted to a temperature of 45℃. The following operations were carried out in a 1 mL glass cuvette: Blank tube: 150uL of distilled water and 850uL of ABTS working solution were added. Sample tube: 150μL of the sample and 850μL of ABTS working solution were added. After thorough mixing, the absorbance value A1 at 10 seconds was measured at 420 nm. The cuvette was quickly placed in a 45℃water bath for 3 minutes, taken out and quickly dried, and the absorbance value A2 at 190 seconds was measured. The calculation ofΔA is as follows:

ΔA (determined tube) = A2 (determined) - A1 (determined)

ΔA (blank tube) = A2 (blank) - A1 (blank)

ΔA = ΔA (determined tube) - ΔA (blank tube)

3. Observation and Recording

During the sample processing, the proportion and operation conditions were strictly followed to ensure good cell disruption and no obvious impurities in the supernatant after centrifugation. In the detection steps, the spectrophotometer was preheated stably, the wavelength was adjusted accurately, and the reading was zero after zeroing with distilled water. The cuvette was mixed evenly after adding reagents, and the time points for measuring absorbance values were accurately grasped. At present, all experimental steps have been completed, and the data processing and laccase activity calculation are awaited.

4. Experimental Analysis

The key to this experiment lies in the accuracy of sample processing and the strict operation of detection steps. The degree of cell disruption will directly affect the release amount of laccase, and the control of temperature, time, and reagent addition amount during the detection process is related to the accuracy of absorbance values. In future experiments, the method of cell disruption can be optimized to improve the extraction efficiency of laccase. At the same time, the operation details in the detection steps can be further standardized to reduce human errors.

5. Experimental Summary

This experiment was carried out smoothly according to the established protocol. Through the sample processing and detection steps, the absorbance value data were obtained. Next, the laccase activity will be calculated according to these data, providing important experimental evidence for subsequent research. During the experiment, the importance of the experimental principle and the necessity of operational norms were deeply understood, which will accumulate valuable experience for future experiments.

##### Experiment 3: Investigation of the Impact of Seawater on the Survival Rate of Recombinant BL21 Escherichia coli

Experimental Dates: 2024/9/18 - 2024/9/23

Experimental Purpose: To study the growth conditions and bactericidal rate of recombinant BL21 Escherichia coli under different conditions.

1. Experimental Steps

1.1 Bacterial Cultivation and Induction

Recombinant BL21 Escherichia coli was placed in a liquid LB medium (pH = 7.0±0.2, containing 100μg/mL ampicillin) and cultivated overnight at 37℃with a rotation speed of 150 rpm.The next day, IPTG with a final concentration of 0.5 mM and Cu2+ with a final concentration of 0.5 mM were added to the culture, and the temperature was adjusted to 30℃ for continued induction cultivation for 16 hours.

1.2 Bacterial Treatment and Counting

The cultured recombinant BL21 Escherichia coli was centrifuged at 2500 rpm for 3 minutes to remove the supernatant. The cells were washed 3 times with LB liquid medium (containing 100μg/mL ampicillin), and then a bacterial suspension was prepared using LB liquid medium. The bacterial suspension was counted using a hemocytometer to make the number of bacteria 1×108 cfu/mL and set aside.

1.3 Experimental Grouping and Treatment

The experiment was divided into 3 groups, namely the experimental group, the positive control group, and the negative control group.

The experimental group: The bacterial suspension was added to an LB culture medium containing a final concentration of 30 g/L sea salt.

The positive control group: The bacterial suspension was added to a normal LB culture medium.

The negative control group: Liquid medium.

The above experiment was repeated 3 times. The lid was closed and gently mixed, and then cultivated for 1 hour.

1.4 Colony Counting and Bactericidal Rate Calculation

The colony counting was carried out by the spread plate method, and the bactericidal rate was calculated.

2、Observation and Recording

During the bacterial cultivation and induction stage, the growth state changes of the culture under different conditions were observed. The bacterial precipitate after centrifugation was obvious, and the purity of the bacteria was ensured during the washing process. After the grouping treatment, there was no obvious difference in the appearance of each group. After 1 hour of cultivation, colony counting was prepared to be carried out, expecting to accurately calculate the bactericidal rate through the counting results.

3、Experimental Analysis

The design of this experiment is relatively rigorous. By using different treatment conditions, the growth conditions and bactericidal effects of recombinant BL21 Escherichia coli in different environments can be compared. During the experiment, strict control of each step of the operation is required to ensure the accuracy of the data. For example, during the counting process, the proper use and accuracy of the hemocytometer should be ensured. For the calculation of the bactericidal rate, the repeatability and error range of the experiment should be considered.

4、Experimental Summary

This experiment was carried out according to the plan, completing the cultivation, treatment, grouping, and preliminary cultivation of bacteria. Next, colony counting and bactericidal rate calculation will be carried out to evaluate the performance of recombinant BL21 Escherichia coli under different conditions. Through this experiment, not only the understanding of bacterial cultivation and experimental design was deepened, but also important reference materials were provided for subsequent research.

##### Experiment 4: Investigation of the Impact of Estradiol Concentration on the Removal Effect

Experimental Dates: 2024/9/24 - 2024/9/28

Experimental Purpose: To study the removal effect of laccase on estradiol in wastewater.

1. Experimental Principle

Laccase can directly catalyze oxygen to oxidize estradiol in wastewater.

2. Experimental Steps

2.1 Experimental Preparation

(1) The starting amount of laccase was set to 7.3 U/mL.

(2) Prepare different starting concentrations of estradiol, which are 0.0005, 0.001, 0.005, 0.01, and 0.05 mmol/L respectively.

(3) A Britton - Robinson buffer solution with pH =5.5 was used as the reaction system, and the total volume of the reaction mixture was 200μL.

2.2 Reaction Process

The reaction system was placed in a constant temperature water bath at 35 °C and reacted for 3 hours.

3. Estradiol Concentration Determination

3.1 Principle of Estradiol Determination

In this experiment, a competitive inhibition enzyme-linked immunosorbent assay is used. The monoclonal antibody of E2 is pre-coated on the enzyme-labeled plate to form a solid-phase carrier. The enzyme-labeled antigen and the antigen to be tested (standard or sample) are simultaneously added to the antibody-coated microplate wells. The antigen to be tested and the enzyme-labeled antigen compete for binding to the specific antibody. After incubation, unbound substances are removed by washing. The amount of bound enzyme antigen is inversely proportional to the concentration of E2 in the sample. The enzyme conjugate is added to the microplate wells, and the content of E2 in the sample is negatively correlated with the color intensity of TMB.

3.2 Steps for Estradiol Determination

(1) The microplate strips to be used were removed from the plate frame, and the remaining strips were put back into the aluminum foil bag containing desiccants and then resealed.

(2) 350μL of 1x washing buffer was added to each well, left to stand for 40 seconds, and then the liquid was discarded. This step was repeated 3 times.

(3) The biotinylated antigen (100x) working solution was prepared 15 minutes before use.

(4) 50μL of standard/sample diluent (R1) was added to the blank well, and different concentrations of standard substances and test samples were added to the other wells, and then 50 μL of biotinylated antigen was immediately added to each well and a new sealing film was covered. The wells were incubated at 37℃ for 1.5 hours.

(5) The streptavidin - HRP (100x) working solution was prepared 15 minutes before use.

(6) The liquid in the wells was discarded, and the washing step in step 2 was repeated.

(7) 100 μL of streptavidin - HRP working solution was added to each well and a new sealing film was covered. The wells were incubated at 37℃ for 30 minutes.

(8) The enzyme reader was preheated.

(9) The liquid in the wells was discarded, and each well was washed with 350 μL of washing liquid, soaked for 1-2 minutes, and the plate was washed repeatedly for 5 times.

(10) 90 μL of TMB substrate was added to each well. The wells were incubated at 37 ℃for 15 - 20 minutes in the dark.

(11) 50 μL of termination liquid was added to each well and immediately placed in the enzyme reader. The OD value at 450 nm of each well was measured within 5 minutes, a correction wavelength was selected and set to 630 nm. The OD value at 630 nm was subtracted from the OD value at 450 nm to correct and remove the OD value of non - coloring substances and obtain more accurate detection results.

4. Observation and Recording

During the experimental preparation stage, laccase and different concentrations of estradiol were accurately configured to ensure the accuracy of the reaction system. During the reaction process, the temperature stability of the constant temperature water bath was closely observed. During the estradiol concentration determination step, the operation flow was strictly followed, paying attention to the time control and reagent addition amount of each step. At present, all experimental steps have been completed, waiting for the detection results of the enzyme reader.

5. Experimental Analysis

The key to this experiment lies in precisely controlling the experimental conditions and operation flow to ensure the accuracy of estradiol concentration determination. Different starting concentrations of estradiol will provide rich data for studying the removal effect of laccase. During the experiment, attention should be paid to the storage and use period of reagents, as well as the aseptic requirements during the operation process to avoid contamination affecting the experimental results. For the use of the enzyme reader, it is necessary to ensure sufficient preheating and correct parameter setting to obtain accurate OD values.

6. Experimental Summary

This experiment was carried out according to the established protocol, through the reaction of laccase with different concentrations of estradiol and the subsequent concentration determination steps, providing important data support for studying the removal effect of laccase on estradiol in wastewater. Next, the detection results will be analyzed, the removal rate will be calculated, and further exploration of the application potential of laccase in wastewater treatment will be carried out.

##### Experiment 5: Investigation of Laccase Production by Recombinant BL21 Escherichia coli on PE Film

Experimental Dates: 2024/9/29 - 2024/9/30

Experimental Purpose: To study the laccase activity and its effect on estradiol under different cultivation conditions.

1. Experimental Steps

1.1 Bacterial Cultivation and Induction

Pick a single colony on the medium into the liquid LB medium (containing 100 μg/mL ampicillin).Cultivate it overnight under the condition of 37℃ and 150 rpm. The next day, add IPTG with a final concentration of 0.5 mM and Cu2+ with a final concentration of 0.5 mM.

1.2 Grouping Cultivation and Treatment

A part of the culture was transferred to a culture dish containing a PE film, with the culture without a PE film as the control group. It was continued to be induced and cultivated at 30℃ for 16 hours.

1.3 Laccase Activity Detection

The laccase activity was detected by taking the culture medium respectively.

1.4 PE Film Group Further Treatment

The PE film group was placed on ice and irradiated with an ultraviolet lamp for 1 hour. Laccase extraction liquid was added, a part of which was used to detect the laccase activity according to the laccase activity detection method, and a part was placed in an estradiol solution with a final concentration of 136.19 ng/mL.

1.5 Estradiol Detection

The reaction was carried out for 3 hours at 35℃, and then the estradiol detection was carried out according to the estradiol content determination method.

2. Observation and Recording

During the bacterial cultivation stage, it was observed that the culture medium gradually became turbid, indicating good bacterial growth. After adding the inducer, the changes in the culture were continued to be observed. During the grouping cultivation, attention was paid to distinguishing the culture dishes with and without a PE film. During the laccase activity detection, the detection results of different groups were recorded. During the PE film group further treatment, the ultraviolet lamp irradiation time and temperature were strictly controlled. During the reaction in the estradiol solution, the time and temperature changes were closely observed.

3. Experimental Analysis

This experiment was designed with multiple steps and comparison groups, aiming to comprehensively study the impact of different conditions on lacc as e activity and estradiol treatment. During the experiment, it is necessary to ensure the accuracy and reproducibility of each step. For the culture dishes containing a PE film, it is necessary to further analyze the mechanism of the impact of the PE film on bacterial growth and laccase activity. At the same time, the ultraviolet lamp irradiation and the addition of laccase extraction liquid may also have an important impact on the experimental results, and it is necessary to explore their principles and effects in depth.

4. Experimental Summary/p>

This experiment was carried out according to the plan, completing the bacterial cultivation, induction, grouping treatment, laccase activity detection and estradiol detection of some steps. Through these experiments, rich data and observation results were obtained, providing important basis for subsequent analysis and research. In the future experiments, the experimental conditions can be further optimized, and the mechanism and application prospects of laccase can be studied in depth.

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# Experiments & Protocol

### Content

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[Overview](#section1)
[Recombinantion](#section2)
[Activity Detection](#section3)
[Seawater](#section4)
[Removal Effect](#section5)
[PE Film](#section6)

# Experiments

### Overview

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Due to the shortage in man power, we give up processing the construction o plasmid, extraction of target DNA and Nucleic acid sequencing experiments. We contracted these experiments to Yunzhou Biotechnology Co., Ltd.

In this project, we do not assign experiments in genetic engineering aspect: we merely accomplish the experiment for characterization which verify the success of build of engineered E.coli.

# Protocols

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### Laccase produced by recombinant BL21 Escherichia coli degrades estradiol

##### 1. Culture of recombinant BL21 Escherichia coli

A single colony on the medium was picked into a liquid LB medium (containing 100 μg/mL ampicillin) and cultured overnight at 37℃ and 150 rpm. The next day, IPTG with a final concentration of 0.5 mM and Cu2+ with a final concentration of 0.5 mM, which had been filtered through a 0.45 μm filter membrane, were added, and the culture was continued at 30℃ for 16 h to obtain the best culture effect.

##### 2. Detection of laccase enzyme activity

2.1 Experimental principle

Laccase decomposes the substrate ABTS to produce ABTS free radicals. The absorption coefficient at 420 nm is much larger than that of the substrate ABTS. By measuring the increasing rate of ABTS free radicals, the laccase activity can be calculated.

2.2 Experimental steps

2.2.1 Sample treatment

According to the ratio of the number of bacteria (108 per mL) to the volume of the extraction liquid (mL) being 20:1, the cells were sonicated in an ice bath (power 300 W, sonication for 3 seconds, interval of 7 seconds, total time of 3 minutes); then centrifuged at 10,000 g for 10 minutes at 4℃, and the supernatant was taken and placed on ice for testing; the culture medium was directly detected.

2.2.2 Detection steps

(1) The spectrophotometer was preheated for more than 30 minutes, the wavelength was adjusted to 420 nm, and zeroed with distilled water.

(2) The temperature of the water bath was adjusted to 45℃.

(3) The following reagents were added into a 1 mL glass cuvette respectively: blank tube: 150 μL distilled water + 850 μL ABTS working solution; sample tube: 150 μL sample + 850 μL ABTS working solution.

(4) After mixing, the absorbance value A1 at 10s was measured at 420 nm. It was quickly placed in a 45℃ water bath for 3 minutes, taken out, quickly dried, and the absorbance value A2 at 190 s was measured. The ΔA of the measurement tube = A2 - A1 of the measurement tube；the ΔA of the blank tube = A2 of the blank tube - A1 of the blank tube, and ΔA = ΔA of the measurement tube - ΔA of the blank tube.

##### 3. Investigation of the effect of seawater on the survival rate of recombinant BL21 Escherichia coli

(1) Recombinant BL21 Escherichia coli was taken into a liquid LB medium (pH = 7.0 ±0.2) (containing 100 μg/mL ampicillin) and cultured overnight at 37℃ and 150 rpm. The next day, IPTG with a final concentration of 0.5 mM and Cu2+ with a final concentration of 0.5 mM were added, and the culture was continued at 30℃ for 16 h.

(2) The cultured recombinant BL21 Escherichia coli was centrifuged at 2500 rpm for 3 minutes, the supernatant was removed, and it was washed 3 times with an LB liquid medium (containing 100 μg/mL ampicillin). A bacterial suspension was prepared with an LB liquid medium; then, the bacterial suspension was counted with a hemocytometer to make the number of bacteria 1×108 cfu/mL and set aside.

(3) The experiment was divided into 3 groups, namely the experimental group, the positive control group, and the negative control group. Experimental group: The bacterial liquid was added to an LB culture medium containing a final concentration of 30 g/L sea salt; positive control group: Normal LB culture medium plus bacterial liquid; negative control group: Liquid medium; The above experiment was repeated 3 times. After covering the lid and gently mixing, the culture was carried out for 1 h, and the colony count was carried out by the spread plate method to calculate the sterilization rate.

##### 4. Investigation of the effect of estradiol concentration on the removal effect

4.1 Experimental principle

Laccase can directly catalyze oxygen to oxidize estradiol in wastewater.

4.2 Experimental steps

A laccase with a starting amount of 7.3U/mL was set, and the starting concentrations of estradiol were 0.0005, 0.001, 0.005, 0.01, 0.05 mmol/L respectively. A Britton - Robinson buffer solution with a pH of 5.5 was used as the reaction system, and the total volume of the reaction mixture was 200 μL. The reaction system was placed in a 35℃ constant temperature water bath for 3 h, then a sample was taken, and the estradiol concentration was measured with an estradiol Elisa kit to calculate the removal rate. The specific detection steps are as follows:

(1) The microporous plate strip to be used was taken down from the plate frame, the remaining plate strips were put back into the aluminum foil bag containing a desiccant, and then resealed for storage.

(2) 350 μL of 1x washing buffer was added to each hole, and the liquid was discarded after standing for 40 seconds. This step was washed 3 times in total.

(3) A biotinylated antigen (100x) working solution was prepared 15 minutes before use.

(4) 50 μL of standard/sample diluent (R1) was added to the blank hole, and different concentrations of standard products and samples to be tested were added to the other holes. Then 50 μL of biotinylated antigen was immediately added to each hole and a new sealing film was covered, and the incubation was carried out at 37℃ for 1.5 hours.

(5) A streptavidin - HRP (100x) working solution was prepared 15 minutes before use.

(6) The liquid in the hole was discarded, and the washing step in step 2 was repeated.

(7) Streptavidin - HRP working solution (100 μL/hole) was added to each hole, and a new sealing film was covered, and the incubation was carried out at 37℃ for 30 minutes.

(8) The enzyme marker was preheated.

(9) The liquid in the hole was discarded, and each hole was washed with 350 μL of washing liquid, soaked for 1 - 2 minutes, and the plate was washed 5 times in total.

(10) TMB substrate (90 μL/hole) was added to the hole. Incubation was carried out at 37℃ in the dark for 15 - 20 minutes.

(11) A termination liquid (50 μL/hole) was added, and it was immediately put into the enzyme marker, and the OD value at 450 nm of each hole was measured within 5 minutes. A correction wavelength was selected and set to 630 nm. And the reading at 450 nm was subtracted from the reading at 630 nm. This way can correct and remove the OD value of non - coloring substances to obtain more accurate detection results.

##### 5. Investigation of the amount of laccase produced by recombinant BL21 Escherichia coli on a PE film

A single colony on the medium was picked into a liquid LB medium (containing 100 μg/mL ampicillin) and cultured overnight at 37℃ and 150 rpm. The next day, IPTG with a final concentration of 0.5 mM and Cu2+ with a final concentration of 0.5 mM were added, and the culture was transferred to a culture dish containing a PE film. A group without a PE film was used as a control group, and the culture was continued at 30℃ for 16 h. The culture medium was taken respectively to detect the laccase activity. At the same time, the PE film group was placed on ice, an ultraviolet lamp was turned on for 1 h, then a laccase extraction liquid was added. One part was detected for laccase activity according to the method in experiment 2.2, and one part was placed in an estradiol solution with a final concentration of 136.19 ng/mL, and after a reaction for 3 h at 35℃, the estradiol was detected according to the method in experiment 1.3.2.

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# Overview

### All experiment results will be shown in this page.

### Target Enzyme

Laccase (multicopper oxidase family protein)

### Gene symbol

AMC88\_RS25540

### Gene description

multicopper oxidase domain-containing protein

### Locus tag

AMC88\_RS25540

### Gene type

protein coding

### Organism

Rhizobium phaseoli (strain: R650, biovar: phaseoli, nat-host: Phaseolus vulgaris)

### Lineage

Bacteria; Pseudomonadota; Alphaproteobacteria; Hyphomicrobiales; Rhizobiaceae; Rhizobium/Agrobacterium group; Rhizobium

### Chassis: BL21(DE3)

# Result

### All experiment results will be shown in this page.

##### Diagnostic enzymatic digestion

Analyze the restriction endonuclease sites on the inserted sequence and the vector backbone, select enzymes that can cut out the appropriate bands to digest the plasmid DNA, run the digested DNA on agarose gel electrophoresis, using EtBrsubstitutes to determine the size of the DNA fragments.

![Image 2](https://static.igem.wiki/teams/5386/resutl/1-plasmid.png)

Restriction Enzyme Digestion Analysis Map

ApaLI(1246,497,2438,3426)

![Image 2](https://static.igem.wiki/teams/5386/resutl/2-strip.png)

Agarose gel electrophoresis pictures

Lane：24

##### Sequence alignment overview

![Image 2](https://static.igem.wiki/teams/5386/result/3-cexujieguo.png)

Conclusion: the sequencing and diagnostic enzymatic digestion confirmed that the construction of our engineering E.coli succeeded.

Detect the activity and the estradiol degradation rate of the induced laccase from the bacterial extract

![Image 2](https://static.igem.wiki/teams/5386/result/4-excel.png)

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