iGEM 2022 — Team npu-china

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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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##### CONTENTS * [Research](#research) * [Build](#build) * [Test](#test) * [Re-design](#redesign) * [Re-test](#retest)

## Research Polycyclic aromatic hydrocarbons (PAHs) are a large group of chemicals. They represent an important concern due to their widespread distribution in the environment, their resistance to biodegradation, their potential to bioaccumulate and their harmful effects [1, 2]. As a promising option, fungal enzymes are regarded as a powerful choice for degradation of PAHs [3]. Phanerochaete chrysosporium, Pleurotus ostreatus and Bjerkandera adusta are most commonly used for the degradation of such compounds due to their production of ligninolytic enzymes. The ligninolytic enzymes are one of the most-efficient oxidative systems found in nature, playing a pivotal role during wood decay and coal formation [4]. The major groups of ligninolytic enzymes include laccases, lignin peroxidases, manganese peroxidases, and versatile peroxidases [5]. At present, bacterial laccase and fungal laccase are more widely used. One study found that laccase CotA from Bacillus subtilis exhibit a higher laccase-specific activity than laccase CueO from E. coli, indicating that CotA is a better candidate for the remediation of PAHs than CueO [6]. Several expression systems have been investigated to produce lignin peroxidases, with fungal hosts that have shown the most promise to date [7]. In our project, laccase CotA from Bacillus subtilis and lignin peroxidase LipH8 from Phanerochaete chrysosporium were overexpressed in E. coli for the remediation of PAHs. [1] O.O. Alegbeleye, B.O. Opeolu, V.A. Jackson, Polycyclic Aromatic Hydrocarbons: A critical review of environmental occurrence and bioremediation, Environmental management, 60 (2017) 758-783. [2] I.A. Titaley, S.L.M. Simonich, M. Larsson, Recent advances in the study of the remediation of polycyclic aromatic compound (PAC)-contaminated soils: transformation products, toxicity, and bioavailability analyses, Environmental science & technology letters, 7 (2020) 873-882. [3] T. Kadri, T. Rouissi, S. Kaur Brar, M. Cledon, S. Sarma, M. Verma, Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by fungal enzymes: A review, Journal of Environmental Sciences, 51 (2017) 52-74. [4] M. Alcalde, Engineering the ligninolytic enzyme consortium, Trends in biotechnology, 33 (2015) 155-162. [5] D.W. Wong, Structure and action mechanism of ligninolytic enzymes, Applied biochemistry and biotechnology, 157 (2009) 174-209. [6] J. Zeng, Q. Zhu, Y. Wu, X. Lin, Oxidation of polycyclic aromatic hydrocarbons using Bacillus subtilis CotA with high laccase activity and copper independence, Chemosphere, 148 (2016) 1-7. [7] O.D.V. Biko, M. Viljoen-Bloom, W.H. van Zyl, Microbial lignin peroxidases: Applications, production challenges and future perspectives, Enzyme and microbial technology, 141 (2020) 109669.

## Build The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli. Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription and translation signals. T7 RNA polymerase is so selective and active that, when fully induced, almost all of the cell's resources are converted to target gene expression. The cotA and lipH8 sequences were cloned into pET28a (+) expression vector, resulting in vectors pHJ5 and pHJ6, respectively. Then the recombinant plasmids were transformed into BL21 (DE3) competent cells, resulting in mutant strains AE5 and AE6, respectively (Fig. 1). The cotA gene was cloned from the genome of Bacillus subtilis, and the lipH8 gene was synthesized by the Sangon Biotech (Shanghai, China). Codon preference or codon usage bias differs in each organism, and it can create challenges for expressing recombinant proteins in heterologous expression systems, resulting in low and unreliable expression. Therefore, we performed gene optimization before synthesizing the lipH8 gene. ![](https://static.igem.wiki/teams/4309/wiki/project/engineering/engineering6.png)Figure 1. Photographs of mutant strains AE5 (a) and AE6 (b) grown on Luria-Bertani (LB) agar plates with kanamycin-50 ug/ml.

## Test The genetic engineering bacteria was constructed and induced by IPTG. In LB medium with adequate nutrition, we conducted induction expression of CotA and LipH8 proteins at different temperatures and times to observe the distribution of proteins. When the OD600 is between 0.6-0.8, it is time to induce the bacteria to produce CotA (Fig. 2) and LipH8 (Fig. 3) with 0.1 mM IPTG. ![](https://static.igem.wiki/teams/4309/wiki/project/engineering/engineering4.png) ![](https://static.igem.wiki/teams/4309/wiki/project/engineering/engineering5.png) Figure 2. SDS-PAGE analysis of AE5 mutant strain CotA. SDS-PAGE was used to analyze the expression of CotA. Recombinant vectors pHJ5 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG for 7 h and 18 h at 16 ℃, 20 ℃, 25 ℃, and 30 ℃. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 1-8, supernatant and pellet fraction of the whole bacterial lysate. S: Supernant; P: Pellet. ![](https://static.igem.wiki/teams/4309/wiki/project/engineering/engineering7.png) Figure 3. SDS-PAGE analysis of AE6 mutant strain LipH8. SDS-PAGE was used to analyze the expression of LipH8. Recombinant vectors pHJ5 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG for 7 h and 18 h at 20 ℃ and 25 ℃. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 1-8, supernatant and pellet fraction of the whole bacterial lysate. S: Supernant; P: Pellet. After several rounds of induction and expression, we found that the protein expressed by CotA was mostly distributed in the supernatant at 16 ℃ and 20 ℃, while the protein expressed by LipH8 was well distributed in the supernatant at 20 ℃ in LB medium. We finally chose 20 ℃ as inducing temperature. ![](https://static.igem.wiki/teams/4309/wiki/project/engineering/engineering1.png) Figure 4. SDS-PAGE analysis of AE5 mutant strain CotA and AE6 mutant strain LipH8. SDS-PAGE was used to analyze the expression of CotA and LipH8. Recombinant vectors pHJ5 and pHJ6 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG in LB medium for 20 h at 20 ℃, respectively. The pellet was then dissolved in MSM medium with 0.1 mM IPTG for 2 d at 20 ℃. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 1-2, whole bacterial lysate of the E.coli BL21(DE3) contained recombinant pET28a-cotA which was induced. Lane 3-4, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-lipH8 which was induced. Lane 5-6, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-cotA and pET28a-lipH8 which were induced. Lane 7-8, whole bacterial lysate of the E.coli BL21 (DE3) containing empty pET28a. S: Supernant; P: Pellet. In the degradation experiment of phenanthrene, 10 mg/L phenanthrene was added as the sole organic carbon source in MSM medium. The pellet of the genetic engineering bacteria was dissolved in MSM medium with 0.1 mM IPTG and 10 mg/L phenanthrene for 2 d at 20 ℃ after 20 h induction at 20 ℃ in LB medium with 0.1 mM IPTG. Unfortunately, we observed that most of the protein was present in pellet fraction of the whole bacterial lysate after 2 d of induction in MSM medium (Fig. 4). This gives us some ideas about how to make proteins fold successfully instead of forming inclusion bodies.

## Re-design We carefully combed protocol and found that the proteins expressed by CotA were almost distributed in the supernatant at 16 ℃, so we made a concession and allowed CotA gene to be induced and expressed at 16 ℃. In other experimental groups, the expression was still induced at 20 ℃. We also noted that the high concentration of IPTG was also the main condition for the formation of inclusion bodies, so we did not add IPTG to the MSM medium. In addition, we heated the engineered bacteria prior to the addition of IPTG to produce heat shock proteins to help the target proteins fold successfully.

## Re-test![](https://static.igem.wiki/teams/4309/wiki/project/engineering/engineering3.png) Figure 5. SDS-PAGE analysis of AE5 mutant strain CotA and AE6 mutant strain LipH8. SDS-PAGE was used to analyze the expression of CotA and LipH8. Recombinant vectors pHJ5 and pHJ6 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG in LB medium for 20 h at 16 ℃ and 20 ℃, respectively. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 1-2, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-cotA which was induced. Lane 3-4, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-lipH8 which was induced. Lane 5-6, whole bacterial lysate of the E.coli BL21 (DE3) containing empty pET28a. S: Supernant; P: Pellet. We followed the idea of Re-Design and changed the temperature and the concentration of the inducer. The figure above clearly shows that the target protein induced in LB medium is present in the supernatant (Fig. 5). This result was encouraging, and we then completed the induction of expression in MSM medium. ![](https://static.igem.wiki/teams/4309/wiki/project/engineering/engineering2.png) Figure 6. SDS-PAGE analysis of AE5 mutant strain CotA and AE6 mutant strain LipH8. SDS-PAGE was used to analyze the expression of CotA and LipH8. Recombinant vectors pHJ5 and pHJ6 transformed into BL21 (DE3) competent cells and induced by 0.1 mM IPTG in LB medium for 20 h at 16 ℃ and 20 ℃, respectively. The pellet was then dissolved in MSM medium without IPTG for 2 d at 20 ℃. All the samples were analyzed by SDS-PAGE, and the protein was stained with Coomassie Blue in the gel. Lane M, protein marker. Lane 1-2, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-cotA which was induced. Lane 3-4, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-lipH8 which was induced. Lane 5-6, whole bacterial lysate of the E.coli BL21 (DE3) contained recombinant pET28a-cotA and pET28a-lipH8 which were induced. Lane 7-8, whole bacterial lysate of the E.coli BL21 (DE3) containing empty pET28a. S: Supernant; P: Pellet. After 2 d of induction in MSM medium without IPTG, the target proteins CotA and LipH8 can be clearly visualized in supernatant fraction of the whole bacterial lysate, which proves that the engineering iteration is effective (Fig. 6). ![](https://static.igem.wiki/teams/4309/wiki/project/engineering/engineering8.png) Figure 7. Oxidation of phenanthrene with the whole bacteria of CotA and LipH8 at 20 ℃ for 1 d, 3d, and 5d. The experiment was carried out in MSM medium without IPTG, and the oxidation was determined using noncellular components as the control. The differences in the PAH oxidation were determined by comparing the controls based on one-way ANOVA followed by Dunnett’s test (\* P < 0.05). SDS-PAGE results showed that the constructed expression system was successful. In order to verify whether the protein had biological activity, the concentration of phenanthrene was detected by HPLC. Both CotA and LipH8 could degrade phenanthrene, and coexpression of CotA and LipH8 was more effective (Fig. 7).

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##### CONTENTS * [Materials and methods](#team_member) + [Construction of Plasmids and Strain](#overview) + [Growth and induction conditions](#gene) + [Protein gel and SDS-PAGE analysis](#suicide) + [Oxidation of PAHs by recombinant CotA and LipH8](#substrate) + [Statistical analysis](#analysis)

# Materials and methods

## Construction of Plasmids and Strain The sequences of all the primers used in this study are listed in Table 1. The strains used in this study are listed in Table 2. To construct pHJ5, the vector pET28a was amplified with the primers pET28a-cotA-F and pET28a-cotA-R, and the 5369 bp PCR products were purified on a gel using the QIAquick gel extraction kit (Qiagen, Germany). The cotA gene (BBa\_K4309000) was amplified from the genome of the wild-type Bacillus subtilis using primers cotA-pET28a-F and cotA-pET28a-R, which included BamHI and SacI restriction sites, respectively. The cotA gene fragment was cloned into the linearized plasmid pET28a using the ClonExpress II one step cloning kit (Vazyme, China), resulting in the plasmid pHJ5. Recombinant vector pHJ5 was transformed into BL21 (DE3) competent cells, resulting in the AE5 mutant strain. The lipH8 gene (BBa\_K4309001) from Phanerochaete chrysosporium was synthesized after gene optimization by Sangon Biotech (Shanghai, China) and cloned into the vector pET28a, resulting in the plasmid pHJ6. The relE gene (BBa\_K4309002) from Escherichia coli was synthesized by Tsingke Biotech (Nanjing, China) and cloned into the vector pBAD43, resulting in the plasmid pHJ9. Recombinant vectors pHJ6 and pHJ9 were transformed into BL21 (DE3) competent cells, resulting in the AE6 and AE9 mutant strains, respectively. Table 1 Primers used in this study![](https://static.igem.wiki/teams/4309/wiki/project/protocol/pro1.png)F, forword; R, reverse. Bold underlined letters indicate restriction sites.Table 2 Strains used in this study![](https://static.igem.wiki/teams/4309/wiki/project/protocol/pro2.png)

## Growth and induction conditions All mutant strain cells were first grown in LB medium (10 g/L trypton, 5 g/L yeast extract, 5 g/L NaCl) with corresponding antibiotics for 3-4 h. Afterwards, the cell suspension was diluted to 1:100 in LB medium with 0.1 mM IPTG. Cultivation for CotA and LipH8 expression analysis was conducted under different induction temperatures and times. Then, this preculture was washed once with MSM medium (5g/L NaCl, 1g/L NH4NO3, 1g/L K2HPO4, 1g/L KH2PO4, 0.2g/L MgSO4∙7H2O, 20 mg/L CaCl2∙2H2O, 5 mg/L FeSO4∙7H2O, 20 mg/L Yeast Extract) without carbon source and transferred into fresh minimal medium to an optical density at 600 nm (OD600) of 0.2. Then, the cell suspension was harvested by centrifugation at 6,000 g for 5 min. The harvested cell precipitation was used for protein expression analysis by SDS-PAGE.

## Protein gel and SDS-PAGE analysis 30 mL of samples were harvested by centrifugation at 6,000 g for 5 min and resuspended in 5 mL of 40 mM Tris-HCl (pH 8.0) and 1 mM phenylmethylsulfonyl fluoride (PMSF) solution (Beyotime, China). The resuspended cells were broken using sonication cell disrupt (Scientz, China) for 10 min on ice (3 s on, 3 s off). The whole-cell lysate was centrifuged at 12,000 g for 10 min at 4 ℃ to remove the cell debris. Insoluble cellular material was removed by centrifugation at 12,000 g for 10 min at 4 °C to divide the supernatant and pellet fraction. The protein concentration was assayed with a BCA protein quantification kit (Solarbio, China). Five micrograms protein with 1X SDS-PAGE sample loading buffer (Baygene, China) per sample were separated by 10% SDS-PAGE and the gel was either stained with 0.1% Coomassie brilliant blue R250 solution in 25% (v/v) isopropanol and 10% (v/v) glacial acetic acid.

## Oxidation of PAHs by recombinant CotA and LipH8 Typical PAH substrates of laccase and lignin peroxidase, phenanthrene were used to investigate PAH oxidation under different conditions, including (1) pET28a only, (2) recombinant CotA only, (3) recombinant LipH8 only, and (4) a combination of recombinant CotA and recombinant LipH8 (0.5:0.5, v/v). For PAHs oxidation analysis, cells were grown in LB medium with 0.1 mM IPTG for 20 h at 16 and 20 ℃ to induce protein CotA and LipH8 expression, respectively. Then, this preculture was washed once with MSM medium without carbon source and transferred into fresh minimal medium containing 10 mg/L phenanthrene to an optical density at 600 nm (OD600) of 0.2. The PAH oxidation reactions were performed in 50 mL Erlenmeyer flasks containing 20 mL of MSM medium with 10 mg/L phenanthrene. The reaction Erlenmeyer flasks were incubated at 20 ℃. After the incubation, the reaction was terminated by adding 50% acetonitrile to a final concentration of 5 mg/L. An aliquot (1 mL) of each sample was centrifuged at 10,000 g for 10 min, and the supernatant was analyzed by high performance liquid chromatography (#LC100, Wufeng Instrument, China). The control strain with pET28a served as the control, and all treatments including the controls were carried out in triplicate.

## Statistical analysis Prism v8.0 (GraphPad Software, USA) was used to process the data, which is the mean ± standard deviation (SD) of three replicates. Statistical differences between the control and treatment groups were determined by one-way ANOVA followed by Dunnett’s test. P < 0.05 indicated statistically significant differences between treatment means and were represented by one asterisk.

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{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## What should this page contain?

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* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

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* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

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You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

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* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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