iGEM 2024 — Team npu-china

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{% block title %}Notebook{% endblock %}
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# Notebook

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* [##### Experimental and Humanistic Practice Notes Overview](#one)
* [##### Experimental Notes](#two)

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## **Experimental and Humanistic Practice Notes Overview**

- Recruiting new team members to bolster our ranks.

- Organizing environmental conservation events to promote sustainability.

- Conducting experiments on lignin degradation to advance biodegradation research.

- Engineering plasmids to enhance genetic manipulation capabilities.

- Refining culture conditions to optimize the efficiency of lignin degradation.

- Engaging in academic conferences and competitions to foster knowledge exchange.

- Presenting the outcomes of our iGEM projects at scholarly gatherings to disseminate our findings.

- Through these initiatives, we have not only expanded our collective knowledge but also contributed to the scientific community's ongoing pursuit of innovation and sustainability.

## **Experimental Notes**

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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# Engineering Success

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* [##### Cycle 1](#cycle1)
* [##### Cycle 2](#cycle2)

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## **Overview**

To realize the goal of advancing the project smoothly, we follow the rule of “Design-Build-Test-Learn Cycle”. We have conducted two-cycles processes to move the project forward. In the first cycle, we overexpressed three genes related to manganese oxidation into plasmid, and finished building and testing the porduction of enzymes (Multicopper oxidase (MCO), Manganese catalase (MnC) and Manganese peroxidase (MnP)) in *P. putida* chassis. In the second cycle, we engineered the Cytochrome c maturation (Ccm) operon with a stronger promoter, achieved to express multiple enzymes plus corresponding manganese oxidation reaction in *P. putida*.

![](https://static.igem.wiki/teams/5204/engineering-success/0.png)

Figure 1: Engineering cycle diagram of our project

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## **Cycle 1-Design**

First, we decided to construct and validate three genes related to Mn oxidation in *P. putida*. Through gene synthesis, we obtained the target gene, cloned the genes of three related enzymes into pBBR1-MCS5 plasmid. Then transformed and tried to express the related enzymes in *P. putida*.

## **Cycle 1-Build**

![](https://static.igem.wiki/teams/5204/engineering-success/1.png)

Figure 2: Colony PCR results for all our samples

## **Cycle 1-Test**

![](https://static.igem.wiki/teams/5204/engineering-success/2.png)

Figure 3: PCR and SDS-PAGE protein check gel electrophoresis result of MCO, MnC and MnP.

                      Control                  MCO                 MnC                  MnP

![](https://static.igem.wiki/teams/5204/engineering-success/3.png)

Figure 4. The Mn oxidation induced by *P. putida*, which overexpresses MCO, MnC and MnP.

As can be seen in Figure 4, the overexpression of each of three enzymes did not achieve the desired manganese oxidation results.

---

## **Cycle 2-Learn**

Because the achieved results is far from satisfatory, we need to find a more efficient way to upregulate the Mn oxidation, thus improving the degradation of lignin. Through learning from literatures and discussions with team advisorss, we learned that the production of enzymes can be achieved by genetic engineering of the promoter of Ccm operon.The promoter can simultaneously regulate the expression of different Ccm enzymes (ccmA, ccmB, ccmC, ccmD, ccmE, ccmF, ccmG, ccmH and ccmI). Therefore, we decided to replaced the promoter of Ccm operon with a stronger one, as shown in our design disgram.

## **Cycle 2-Design**

We use a stronger promoter (part code: BBa\_5204002), which is reported to be a stronger promotor of *P. putida*, to replace the original promoter of Ccm operon. Then we constructed the vectorin *P. putida* In addition,gentamicin was added to all media to ensure that the surviving cells contained our construction plasmid.

![](https://static.igem.wiki/teams/5204/engineering-success/4.png)

Figure 5. Schematic representation of the ccm operon of Pseudomonas putida.

## **Cycle 2-Build**

![](https://static.igem.wiki/teams/5204/engineering-success/8.png)

Figure 6. Promoter modification engineering to improve the transcript level of the ccm operon.

## **Cycle 2-Test**

![](https://static.igem.wiki/teams/5204/engineering-success/5.png)

Figure 7: QPCR results of ccmE and ccmL, and the Mn oxidation results induced by *P. putida*.

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{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# Experiments

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* [##### The cloning and expression of target genes](#The-cloning-and-expression-of-target-genes)
* [##### Isolation, purification and activity identification of the target protein](#Isolation, purification-and-activity-identification-of-the-target-protein)
* [##### The starter transformation of the target operator](#The-starter-transformation-of-the-target-operator)
* [##### Biosynthesis of manganese oxides](#Biosynthesis-of-manganese-oxides)
* [##### Degradation and utilisation of lignocellulose](#Degradation-and-utilisation-of-lignocellulose)

---

## **Experiments Overview**

This experiment page includes the protocol we used in the wet lab experiments, from obtaining the target fragment to build the plasmid, to transforming the constructed plasmid into *P. putida*, finally by systematic characterizaiton of the structure of manganese oxide, and the degradation performance of lignin.

---

## **Process**

### **1. The cloning and expression of target genes**

#### 1.1 Target gene sequence search

Alternative CumA, source: Pseudomonas putida. On the NCBI website, the GenBank database was utilized to search for sequences. Click on "download datasets" to download gene sequences, protein sequences, and other information.

#### 1.2 Primer design and synthesis

Primer5 was used to design primers.

#### 1.3 Amplification of the target gene

1.3.1 Genome DNA was extracted from the cells

Lysozyme was added, and the sample was placed in a water bath for complete lysis (cell fragmentation). Nucleic acids were extracted by fragmentation, and impurities were removed. The resulting nucleic acids were purified.

1.3.2 PCR was used to amplify the known gene fragments

PCR amplification was performed using the PCR in vitro amplification technique with a PCR apparatus. A 0.2mL enzyme-free PCR reaction tube was used to add each reagent separately with a microsample gun in the order described below.

Table 1 Amplification PCR reagent and dosage

![](https://static.igem.wiki/teams/5204/experiments/0.png)

#### 1.4 Gel electrophoresis, recovery and purification of DNA

1.4.1 Gel electrophoresis of DNA

Rinse the gel mold and comb with distilled water, place them on the gel plate, and secure the comb. Prepare the gel. Once the gel has completely set at room temperature, carefully remove the comb and place the gel into the electrophoresis tank. Pour the electrophoresis buffer into the tank, ensuring the buffer level is 1mm below the gel surface. Remove any bubbles in the sample wells. After adding loading buffer to the DNA sample, slowly introduce the mixture into the submerged gel loading port with a pipette. Turn on the power, with red indicating the positive electrode and black the negative electrode. Ensure DNA samples migrate from the negative to the positive electrode (add one end close to the sample well). Typically, a voltage of 50V is used for electrophoresis, which can last for 90 minutes. Terminate electrophoresis based on the migration of the indicator dye. Post-electrophoresis, turn off the power, observe using nucleic acid dye staining and an ultraviolet detector, and record the electrophoresis results.

1.4.2 Recovery and purification of PCR products from gels

Under ultraviolet light, cut out agarose gel blocks containing the target DNA fragments. Weigh the gel block and add sol solution according to a specific ratio. Heat the sol in a 55℃ water bath, gently mixing until the gel is completely dissolved. Transfer the dissolved solution to an adsorption column and centrifuge to separate the DNA. Wash away impurities with a wash solution, and finally elute the DNA with the eluate to obtain purified PCR products. Store as instructed.

#### 1.5 Double enzyme digestion, recovery and purification of the target gene

1.5.1 Double enzyme digestion of the target gene

Add the following reagents in proportion to a double digestion system in sterile centrifuge tubes.

Table 2 Double enzyme digestion reaction system

![](https://static.igem.wiki/teams/5204/experiments/1.png)

1.5.2 Recovery and purification of restriction enzyme products

Estimate the relative amount of recovered target genes by AGAR gel electrophoresis and purification using a nucleic acid quantification apparatus.

#### 1.6 Double enzyme digestion, recovery and purification of the expression vector

1.6.1 Double restriction enzyme digestion of the expression vector

Add the following reagents in proportion to a double digestion system in sterile centrifuge tubes.

Table 3 Double enzyme digestion reaction system

![](https://static.igem.wiki/teams/5204/experiments/2.png)

1.6.2 Recovery and purification of restriction enzyme products

Detect the digested vector by AGAR gel electrophoresis and recover and purify using a vector extraction kit.

#### 1.7 The target gene was linked to the expression vector

Prepare the ligation system by adding the following reagents in proportion to a sterile centrifuge tube (the ratio of control expression vector to target gene should be 1:3-1:10; Ligate at 4℃ for 1 hour and store at 4℃).

Table 4 Connection reaction system

![](https://static.igem.wiki/teams/5204/experiments/3.png)

#### 1.8 Preparation and transformation of competent cells

1.8.1 Preparation of competent cells

Add single colonies to 5 mL LB liquid medium and incubate overnight at 37 ℃ at 180rpm with shaking. Inoculate 5% (2.5 mL) bacterial solution into a shaking flask containing 50mL LB and incubate at 37 ℃ for 2-3 hours to OD600 = 0.4-0.5. Place cultures in an ice water bath for 20 minutes. Collect cells by centrifugation at 5000rpm and 4℃ for 5 minutes. Discard the culture solution, trying to remove as much of it as possible. Suspend cells in 20 mL of 0.1 mol/L CaCl2 precooled in an ice bath for 20 minutes. Centrifuge at 5000 rpm for 5 minutes at 4℃ and decant the supernatant. Suspend cells again with 10mL of 0.1mol/L CaCl2 precooled, then centrifuge. Prepare competent cells by adding 2 mL of 0.1 mol/L CaCl2 precooled and gently suspending the cells. They can be used immediately or divided into 200 µL aliquots for future use and frozen at -80 ℃.

1.8.2 Transformation of plasmids

Transform the ligation products of the target gene and the expression vector into competent cells using an ice bath and heat shock method. Then, spread the cells on the surface of LB medium containing gentamicin and culture in an incubator at 30 ℃ for 12 hours. Set up a positive control group and a negative control group for comparative observation.

#### 1.9 Identification of monoclonal colony broth by PCR

1.9.1 Enrichment cultivation

Transfer single colonies selected from the plates to 5 mL of liquid LB medium containing 1% gentamicin and incubate overnight at 30 ℃ at 200rpm.

1.9.2 Bacterial fluid was identified by PCR

Amplify the target gene fragment by PCR using bacterial broth as a template and verify the length of the target gene by agarose gel electrophoresis. The operating system is as follows:

Table5 Reagent and dosage of PCR amplification

![](https://static.igem.wiki/teams/5204/experiments/4.png)

#### 1.10 Extraction, purification and identification of recombinant plasmids

1.10.1 Extraction of recombinant plasmids

Extract recombinant plasmids from Pseudomonas putida using a vector extraction kit. Digest the plasmids with single and double restriction enzymes, respectively. Preliminary identification of target genes is done by gel electrophoresis.

1.10.2 Sequence identification of recombinant plasmids

Once the above identification results are correct, send the recombinant plasmid to a company for sequence identification.

---

### **2. Isolation, purification and activity identification of the target protein**

#### 2.1 Expanded culture of engineering bacteria

Carry out the scale-up culture in a clean bench. Store the obtained bacterial solution and transfer 5 μL of seed solution to 5 mL of LB liquid medium containing gentamicin, incubate overnight at 37 ℃, 220 rpm. Then, transfer to 50 mL of liquid LB medium at a volume ratio of 1:1000.

#### 2.2 Objective Induction and expression of protein

After the bacteria have been cultured for 6 hours, add 500 μL of IPTG to the medium to achieve a final concentration of 0.1 mM for induced expression. Subsequently, transfer the medium to a 30 ℃, 200 rpm shaker for 8 hours.

#### 2.3 Break the ultrasonic pulse of engineering bacteria

Remove the Erlenmeyer flask from the shaker and transfer the bacterial solution to a 50 mL centrifuge tube to collect the bacterial pellet, using centrifugation parameters of (4 ℃, 10000×g, 5 minutes). Wash the bacterial pellet three times with ddH2O, then resuspend the bacteria in 5 mL of PBS buffer. Subsequently, transfer the bacterial suspension to ice and disrupt the bacteria completely using a cell breaker with ultrasonication for 15 minutes (power 60%; Sonication for 3 seconds, intermittent for 3 seconds). Centrifuge the crushing solution at 10,000×g at 4 ℃ for 15 minutes, collect a small amount of supernatant and precipitate separately. Filter the remaining supernatant through a 0.22 μm ultrafiltration membrane and collect the filtrate for affinity chromatography.

#### 2.4 Affinity chromatography to isolate the target protein

2.4.1 Protein purification.

Equilibrate the column using 5 column volumes of equilibration buffer. Load the supernatant of the filtered lysate onto the balanced chromatography column, collecting approximately 1 mL of the lysate supernatant and labeling it for storage. Elute heteroproteins with 10 column volumes of equilibrium buffer, retaining approximately 1 mL of heteroprotein eluent for electrophoresis during the final column volume elution. Elute the target protein using 2 column volumes of elution buffer and collect all eluates. Wash the desalting column using 10 column volumes of 500 mM imidazole elution buffer. Clean the column using 10 column volumes of pure water. Seal the column with 5 column volumes of 20% ethanol and store tightly closed at 4~8 ℃.

2.4.2 Desalination

Clean the desalination column with 3 column volumes of pure water. Equilibrate the chromatography column with 5 column volumes of desalting buffer PB. Load 1.5 mL of the protein solution post-affinity chromatography onto the desalting column, retaining a small amount of eluted protein solution samples. Elute the protein of interest using 2 mL of desalting buffer and collect the protein of interest. Equilibrate the chromatography column with 5 column volumes of desalting buffer. Wash the desalination column using 10 column volumes of pure water. After sealing the column with 5 column volumes of 20% ethanol, seal and store at 4~8 ℃. Store all collected sample solutions at 4 ℃, adding glycerol to the samples to achieve a final concentration of 5% post-desalting and store at 4 ℃.

#### 2.5 SDS-PAGE identification of the target protein

2.5.1 SDS polyacrylamide gel production

Utilize Solarbio's SDS-PAGE Gel Preparation Kit to prepare gels in the experiment. Leak detection: Identify the matching 1.5 mm rubber plate, rinse it well, dry it with lens polishing paper, and secure it on the dispenser. Conduct a leak test with deionized water. Preparation of separating gel: After 5 minutes, if the gel board does not leak, prepare the separating gel. Prepare 10 mL of 12% resolving gel according to the kit's ingredient table (Table 6). Filling and separating the gel: Pour out the water in the rubber board and absorb the water with filter paper. Take 5 mL of the mixed separating gel and gently pour it along the edge of the gel plate to prevent bubbles, and level the separating gel with water. Preparation of stacking gel. Prepare 5 mL of 5% stacking gel according to the kit's ingredient table (Table 7). Filling concentrated gel: A distinct horizontal line forms between the separating gel and the water, indicating that the separating gel has solidified. Pour out the water on the top layer of the separating gel, absorb the water with filter paper, take the mixed concentrated gel and gently pour it along the edge of the rubber plate, insert a comb with 11 wells, and let it stand for 30 minutes. Prepare running buffer (500 mL). Follow the ingredient table of the electrophoresis buffer (Table 8). Turn the gel maker upside down, ensuring the gel does not flow out. Load the solidified gel plate into the electrophoresis tank and add SDS-PAGE electrophoresis buffer to soak the gel for a period.

Table 6. Preparation Method of Separating Gel

![](https://static.igem.wiki/teams/5204/experiments/5.png)

Table 7. Preparation Method of Stacking Gel

![](https://static.igem.wiki/teams/5204/experiments/6.jpg)

Table 8. Reagent Table for 12% Electrophoresis Buffer (1 L)

![](https://static.igem.wiki/teams/5204/experiments/7.png)

2.5.2 Sampling-sampling electrophoresis

Take 10 centrifuge tubes and label the samples as "pre-induction, post-induction, broken supernatant, broken precipitate, penetrating solution 1, penetrating solution 2, 5 μg pre-elution protein, 10 μg pre-eluting protein, 5 μg post-elution protein, 10 μg eluting protein" in the order of "1-1". Mix 40 μL of protein sample with 10 μL of 4× loading buffer in a 1.5 mL centrifuge tube, mix well, and heat in boiling water for 10 minutes. Allow to cool to room temperature. Load 10 μL of sample and 5 μL of protein marker (15-130 KDa). Initiate electrophoresis at a constant voltage of 80 V, increase to 120 V after the samples enter the separating gel, and stop electrophoresis when the blue bromophenol blue band migrates to about 1 cm from the lower end of the gel. Cut off the power, unplug and pour the electrode buffer into a large beaker. Unload: Remove the rubber plate from the electrophoresis instrument, pry the rubber plate open with a blade, and separate the concentrated rubber part of the gel. Staining: Add Coomassie Brilliant Blue to the extracted stacking gel for 15 minutes, then transfer to the decolorization solution for destaining. After the eluent no longer changes color, take a picture and store it.

#### 2.6 Enzymatic detection of the target protein

Conduct the entire assay on ice. Prepare the assay system according to Table 9, prepare 4 samples for each system, and finally add 3 wells.

Table 9. MDH enzyme activity assay system

![](https://static.igem.wiki/teams/5204/experiments/8.png)

Set the microplate reader parameters: temperature 30 ℃, vibrating plate 30 seconds, wavelength 340 nm, kinetics 15 minutes, and 1-minute intervals, so we will have 15 data points for each well of the final sample.Based on the increase in absorbance at a specific wavelength and the molar extinction coefficient of the reaction product, calculate the amount of reaction product generated per minute, which can then be used to further calculate the enzyme activity and specific activity.

---

### **3. The starter transformation of the target operator**

#### 3.1 Target operon sequence search

3.1.1 Obtain the standard name of the target gene or protein by referring to the literature.

3.1.2 Conduct a search on the NCBI website to obtain the input operon sequence.

#### 3.2 Sequence synthesis of constitutive promoters

Constitutive promoters enable gene expression to be activated in all tissues. Conduct a search on the NCBI website to obtain the constitutive promoter of the desired species.

#### 3.3 Integration of the constituent promoter

3.3.1 Synthesis of sgRNA primers using the CRISPR-Cas system

Select the target DNA sequence: identify the gene to be edited and the purpose of editing, such as targeting a specific gene mutation, deletion, or replacement. Determine the target sequence: according to the editing purpose, determine the target sequence that needs to be edited in the target gene, usually a sequence of 20 nucleotides. Design the sgRNA sequence: sgRNA typically consists of two parts: the target sequence and the scaffold sequence. The scaffold sequences usually include transcription initiation sites at the 5' end and termination sites at the 3' end, which are critical for the proper functioning of the CRISPR-Cas9 system. The target sequence should be complementary to the target sequence of the target gene to ensure specificity. Determine the appropriate primer length: the primer length is usually 17-20 nucleotides, and the shorter the primer, the greater the likelihood of non-specific binding and shearing. The selection of length needs to be optimized according to different specific experiments and cell types. Determine the appropriate location of the sgRNA primer: usually, the appropriate location is selected in the promoter or exon region of the target gene. Use online tools for validation: Use online tools or software to verify the specificity and accuracy of sgRNA, such as Crispr design. Synthesis of sgRNA primers: The designed sgRNA sequence is synthesized into RNA molecules.

3.3.2 Construction of sgRNA-Cas9 plasmid and repair of DNA template plasmid

After preparing the sgRNA, the Cas9 protein and sgRNA need to be expressed. Methods such as gene transfection, electroporation, or viral vectors are usually used to introduce Cas9 and sgRNA into cells. These methods ensure that enough Cas9 and sgRNA reach the target gene for effective gene knockout.

3.3.3 The recombination of DNA sequences

Once Cas9 and sgRNA have successfully entered the cell, Cas9 binds to the sgRNA and cuts the DNA sequence of the target gene. Cas9 typically cuts DNA around a primer sequence specified in the sgRNA.

3.3.4 Verifying Successful typing

Purine screening, fluorescence detection, and PCR to detect the genome, RFP protein is knocked in.

#### 3.4 PCR identification of bacterial solution

Take the modified bacterial solution, dilute and spread on the resistant plate to form a monoclonal; use an autoclaved tip to pick a single colony and punch the tip into 5 mL containing... Shake the tube of resistant LB liquid medium and incubate it overnight at 37 ℃; take 1 μL of bacterial solution, add primers, ddH2O, Rapid TaQ, and Master Mix to configure the PCR reaction system, and adjust the PCR instrument program for PCR. Gel, electrophoresis buffer, sample loading, nucleic acid gel electrophoresis to detect PCR products.

#### 3.5 Gene Sequence Identification Sanger Sequencing

PCR amplification of the DNA fragment to be tested; prepare 4 dNTPs and DNA polymerases containing DNA templates, primers, 4 radiolabeled dNTPs, add 4 ddNTPs in proportion, and adjust the instrument to start the reaction; The reaction products in four reaction tubes were separated by denaturing polyacrylamide gel electrophoresis, and the DNA sequence was estimated according to the length of the fragments and the terminal base information.

#### 3.6 Q-PCR identification of downstream gene transcripts

To verify the feasibility of the promoter, the transcription of the downstream ccmE gene was detected by QPCR. First, extract total bacterial RNA using an RNA extraction kit and reverse-transcribe the corresponding cDNA. Subsequently, add buffers, upstream and downstream primers, DNA polymerase, deoxyribonucleic acid, dyes, etc., sequentially, and obtain the amplification curve and dissolution curve using the SYBR-Green PCR instrument. Obtain the expression of the target gene by using the unmodified Pseudomonas putidatum as the control.

---

### **4. Biosynthesis of manganese oxides**

#### 4.1 Activation of seed solution

Seed the Pseudomonas aeruginosa seed solution into 5 mL of LB medium at an inoculum rate of 1% and activate overnight on a constant temperature shaker at 30℃ and 200 rpm.

#### 4.2 Expanded culture of bacteria

Inoculate the activated seed solution into 50 mL of BL medium at a volume ratio of 1:1000 for expanded culture.

#### 4.3 Synthesis of manganese oxides

After 12 hours of further incubation, add 500 mg of manganese sulfate to 50 mL of BL medium as a manganese source to synthesize manganese oxides in co-culture with *P. putida*.

#### 4.4 Isolation and purification of manganese oxides

Collect organisms using a cryogenic high-speed centrifuge at 4000 rpm/min, then break the organisms using an ultrasonic crusher. Collect the precipitates after high-speed centrifugation and wash three times with ddH2O and DMF, followed by freeze-drying.

#### 4.5 Characterisation of manganese oxides

Characterize the resulting precipitates using SEM, XRD, XPS, IR, and other characterization tools.

#### 4.6 Characterisation of enzyme-like activity of manganese oxides

POD-like enzyme activity test: Weigh the obtained manganese oxides and dissolve them by sonication using ddH2O at 2 mg/mL, and weigh 6 mg of TMB and dissolve it in 1 mL of DMSO to obtain a 25 mM solution of TMB. Add 10 μL of manganese oxides solution to 10 μL of acetate buffer pH 3.6 and TMB solution, and measure the UV absorbance at OD652nm. Laccase-like activity test.

Lac-like enzyme activity test: Add 970 μL of MES buffer (0.1 M, pH 6.8) sequentially with nanozymes (2 mg/mL), 10 μL of 2,4-dichlorophenol (2.5 mg/mL), 10 μL of 4-aminoantipyrine (100 μg/mL), followed by measurement of absorbance at 510 nm using Nanodrop one.

---

### **5. Degradation and utilisation of lignocellulose**

#### 5.1 Degradation of lignocellulose

Use native maize stover as a lignocellulose model and add stover powder to ddH2O with the previously obtained manganese oxide nano-enzymes. Carry out the reaction on a magnetic stirring table at room temperature at a speed of 800 rpm.

5.1.1 Concentration change of lignocellulose

Take the supernatant before and after the reaction and measure the absorbance changes at OD280nm and OD355nm using Nanodrop.

5.1.2 Structural changes of lignocellulose

Take the lignin before and after the reaction and freeze-dry. Detect the changes in functional groups of the above samples using Fourier Infrared Spectroscopy.

5.1.3 Morphological changes of lignocellulose

Obtain the morphological changes of lignocellulose before and after the reaction using SEM electron microscopy.

#### 5.2 Utilization of lignocellulose by bacteria

Inoculate Pseudomonas aeruginosa into the medium for cultivation at a volume ratio of 1:1000. Set the medium as: de-glucose sugar M9 medium and lignin as carbon source M9 medium.

5.2.1 Changes in bacterial biomass

Incubate the above medium at 30 ℃, 220 rpm for 12 hours. After 12 hours, spread 100 μL of the culture solution on LB solid medium for incubation. After 12 hours, count the number of bacterial colonies.

5.2.2 Size of bacterial monoclonal colonies

Centrifuge the bacterial culture solution cultured to OD600=0.4, wash, and dilute with ddH2O to OD600=0.2. Then, aspirate 100 microliters of the bacterial solution and spread on solid desugared M9 medium and solid lignin desugared medium, respectively. Culture in an incubator at 30 oC for 48 hours. After 48 hours, photograph and sample the bacterial plates and record the differences in the size of the morphology of the monoclonal colonies.

5.2.3 Manganese oxides assist bacteria to utilize lignin

Transfer a certain number of bacteria to lignin M9 medium without glucose. Set up the following groups: (1) Deglycosylated lignin M9 medium; (2)Bacteria+deglycosylated lignin M9 medium; and (3) Bacteria+manganese oxides+deglycosylated lignin M9 medium. Detect the lignin changes after incubation in the incubator at 30 ℃, 220 rpm for 48 hours. The results are shown in the table below.

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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# Results

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[![](https://static.igem.wiki/teams/5204/description/linker.svg)](#lk1)

* [##### Manganese Oxide Nanozyme Characterization](#Manganese-Oxide-Nanozyme-Characterization)
* [##### Degradation of lignin (dealkaline lignin).](#Degradation-of-lignin)

---

## **Overview**

Our preliminary design and engineering efforts have yielded promising experimental outcomes. The success of our engineering was substantiated by the analysis of the collected precipitate and the subsequent assay of its enzyme-like activity. Ultimately, we deployed our Ccm-promoter bioreactor for the degradation of lignin and corn stalks, achieving a notably enhanced degradation rate.

---

## **Manganese Oxide Nanozyme Characterization**

Our findings indicate that the culture medium supplemented with Mn2+ exhibits a distinct color variation. In a control experiment devoid of bacteria, there is no significant color change observed. However, in the presence of bacteria, the recombinant strain of *P. putida* displays a noticeably deeper brownish-yellow hue compared to the wild type. This coloration is likely attributable to the formation of high-valence manganese oxide (MnOx). Subsequently, the precipitate formed during the cultivation of *P. putida* was harvested.

The structural characteristics of the precipitate were meticulously examined using transmission electron microscopy (TEM) and X-ray diffraction (XRD), and their manganese catalase (MCO)-like activities were evaluated. The TEM images revealed that all samples are nanomaterials with distinct morphologies. Analysis of the XRD spectra indicated that Mn2+ was auto-oxidized to MnO in ambient conditions. The wild type *P. putida* facilitated the formation of Mn2O3, whereas the recombinant strain produced Mn3O4. Based on the activity assays, MnO exhibited negligible enzymatic activity. In contrast, Mn2O3 demonstrated typical enzyme-like activity, albeit significantly inferior to that of Mn3O4.

![](https://static.igem.wiki/teams/5204/results/0.png)

Figure 1. The images of Mn2+ oxidation without bacteria, with *P. putida* and ccm-promoter engineered *P. putida*.

![](https://static.igem.wiki/teams/5204/results/1.png)

Figure 2. The image of MnOx collected from different culture medium in Figure 1.

![](https://static.igem.wiki/teams/5204/results/2.png)

Figure 3. The TEM images of produced MnOx in Figure 2 and their XRD spectra.

![](https://static.igem.wiki/teams/5204/results/3.png)

Figure 4. The XRD spectra of produced MnOx in Figure2.

![](https://static.igem.wiki/teams/5204/results/4.png)

Figure 5. The laccase oxidase-like activity of produced MnOX and corresponding UV-vis absorbance spectra of reaction system.

![](https://static.igem.wiki/teams/5204/results/5.png)

Figure 6. The laccase oxidase-like activity of ccm-promoter engineered *P. putida*.

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## **Degradation of lignin (dealkaline lignin).**

To evaluate our design, the lignin degradation performance of the recombinant *P. putida* bioreactor was tested by culturing it in a dealkaline medium. The concentration, morphology, structural features, and chemical composition were analyzed. Furthermore, *P. putida* was cultured with both raw and treated corn stalks to assess the potential generation of carbon sources following degradation. Collectively, these analyses demonstrated that the ccm-promoter engineered *P. putida* exhibits significantly enhanced degradation capabilities towards lignocellulose, thereby validating our successful engineering efforts. Moreover, the engineered *P. putida* could serve as an excellent platform for the utilization and transformation of lignocellulose, offering a promising pathway for the development of renewable energy sources.

![](https://static.igem.wiki/teams/5204/results/13.png)

Figure 7. The image of lignin-containing solution before and after treatment.

![](https://static.igem.wiki/teams/5204/results/7.png)

Figure 8. The time-dependent UV-vis spectra of dealkaline lignin-contained system.

![](https://static.igem.wiki/teams/5204/results/8.png)

Figure 9. The images of corn stalk before and after degradation.

![](https://static.igem.wiki/teams/5204/results/9.png)

Figure 10. SEM images of corn stalk before and after degradation

![](https://static.igem.wiki/teams/5204/results/14.png)

Figure 11. XRD and FTIR spectra of lignin before and after degradation.

![](https://static.igem.wiki/teams/5204/results/15.png)

Figure 12. The three-dimensional excitation-emission matrix fluorescence spectroscopy of the corn stalk along with reaction time.

![](https://static.igem.wiki/teams/5204/results/12.png)

Figure 13. The plate count results of *P. putida* cultured with raw lignin (a), M9 full medium (b) and treated corn stalk (c), and its corresponding colony count.

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