iGEM 2018 — Team Nanjing-China

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# Team:Nanjing-China/Notebook

Nanjing-China2018

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  + [Design](https://2018.igem.org/Team%3ANanjing-China/Design)
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* [MODELING](https://2018.igem.org/Team%3ANanjing-China/Model)
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* [NOTEBOOK](https://2018.igem.org/Team%3ANanjing-China/Notebook)

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March

3

Our team was founded this week! We met and communicated with each other. Through our team leader’s presentation, we knew iGEM a lot. Each member was assigned his/her mission in the team.

We broadly read paper and brainstormed this year’s project. At first we came up with several different ideas, later we reached an agreement that the most interesting and meaningful one was about nitrogen fixation.

April

4

#### Human Practices, Collaboration&Society:

We learned about the advancement of nitrogenous fertilizer production in China. In order to better understand the actual demand of nitrogenous fertilizer in agriculture, we decided to visit farmers in Xiaohe Bei Village.

We distributed brochures about our project at NJU.

Having learned of the dearth of efficient and affordable fertilizer, we spared no effort to seek a cost effective nitrogen fixation method. Inspired by our previous work(Nanjing-China 2016), we creatively proposed an idea of “whole-cell photocatalytic nitrogen fixation”.

We helped Nanjing Forestry University build their team.

We held conferences with Nanjing Agricultural University and China Pharmaceutical University to share experiences of being iGEMers.

#### Technical works Wet& Dry labs:

Wet Lab: Having confirmed the theme of our project, we began to work on our design. We read latest papers about biological nitrogen fixation, focused on the method sections and discussed what we didn’t understand in details. During the last week of this month, we worked out the first version of our design.

Dry lab: We communicated and exchanged ideas frequently in order to identify possible modeling directions which could provide useful guidance to our wet experiments. Later we proposed a few directions. The idea of developing homologous modeling of nitrogenase didn’t work successfully because we couldn’t get access to relevant software.

May

5

#### Human Practices, Collaboration& Society:

We planned to investigate the current production of nitrogenous fertilizer so we prepared interview questions and contacted Yantai Wuzhou Feng Fertilizer Plant. Then we went there, met the manager and were shown around the factories. We communicated with the technical R&D personnel and realized the big challenge we had to overcome before putting our project into practical application.

#### Technical works Wet& Dry labs:

Wet lab: We measured the transcriptional activity of *nif* promoter. Then we transformed the plasmid pUC57 containing the *nif* cluster and the fusion protein expression plasmid including *E. coli* outer membrane protein *OmpA* and the *PbrR* protein into *E. coli* strain JM109. Besides, based on our Human Practice, we modified our design by adding Cd2+ toxicity test to it.

June

6

#### Exam Break

July

7

#### Human Practices, Collaboration& Society:

We invited Professor Haoqian Zhang and held a meet up with iGEM teams in Nanjing.

We were interviewed by Nanjing University Student Career Guidance Center. The Wechat Push introducing our team was issued on the public account “ NJU Employment”.

We borrowed the No.5 plasmid in the InterLab kit to team AHUT.

#### Technical works Wet& Dry labs:

Wet lab: We conducted Real-time Quantitative PCR(qPCR). Meanwhile, we conducted Cd2+ toxicity test and ICP-MS measurement of Cd2+ adsorption.

We improved our part to make it easier to operate.

Dry lab: Enlightened by the different relative transcriptional levels of each nitrogenase component which was shown in the result of qPCR, we turned our attention to the complexity of nitrogenase system. We perused literature on the stoichiometry of nitrogenase components.

August

8

#### Human Practices, Collaboration& Society:

We attended the 5th Conference of China iGEMer Community at Shanghai Tech University to demonstrate our project to all teams in China and learn from each other.

We helped Central South University found team.

We communicated with two members of iGEM USTC.

#### Technical works Wet& Dry labs:

Wet lab: We conducted TEM-EDX analysis and UV-vis scanning. Then we used methyl viologen to verify the generation of electron. Finally, we successfully biosynthesized CdS semiconductor which could be excited by visible light to generate electrons.

Dry lab: We finally decided to model on the best stoichiometry of *nif* gene cluster. We looked through many common algorithms and figured out two modeling methods. After further comparison, we finally chose a method similar to greedy algorithm. We drew a flow diagram to describe the core idea of our method and as a reference for programming. Then we programmed with python, debugged our code and received the result.

September

9

#### Human Practices, Collaboration& Society:

We performed Language project with IIT Madras and received the finished video a few days later.
We helped another iGEM team, CSU-China to establish their team. We issued our Emoji chanllenge on the official website and received some interesting feedback shortly after that.

#### Technical works Wet& Dry labs:

Wet lab: Inspired by our Human Practice, we raised an idea of designing a device for the growth of engineered *E.coli* strain*.* First we drew a draft on paper and then used the software Solid Works to draw a 3D version draft. Eventually, a real device came out. The device provided a great help to our further experiments because it provided a suitable place for the engineered strain to grow.

Near the end of this month, we performed gas chromatography to detect the amount of acetylene reduced to indirectly test the nitrogen fixation activity of our system. Up to wiki freeze, samples have been sent out and we will receive the result about five days later.

Dry lab: We refined our model by further reading literature and verifying one of our assumptions. In that way, we got a more accurate result. This result provided useful guidance to our further experiments.

## Protocol

### Plasmids and Bacterial Strains.

The bacterial strains, plasmids and primers used in this study are all listed in Table 1. *Escherichia coli*JM109 was purchased from Takara and designated EJ. A high-copy plasmid, pUC57-*nif* (pMB1 *ori*), harboring the minimal nitrogen fixation gene cluster (*nif*) of *Paenibacillus polymyxa* CR1 was chemically synthesized and then transformed into *E. coli* JM109, and the resulting recombinant was designated EJN. For construction of the second plasmid, pJQ200SK *OmpA/PbrR* (with a compatible p15A *ori*), a lab store plasmid pBAD24-*OmpA/PbrR* was used as the template to PCR-amplify *OmpA/PbrR* with P200F and P200R primers. After confirmation by sequencing, the PCR product was digested with *Kpn* I and *Hind* III and then insert into pJQ200SK to yield pJQ200SK-*OmpA/PbrR*. EJN transformed with pJQ200SK-*OmpA/PbrR* was selected from LB agar plates containing appropriate antibiotics, and the resulting strain was designated EJNC.

### Culture Conditions.

LB broth for *E. coli* JM109 growth contained 10g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract. KPM minimal medium was adopted for all nitrogen fixation assays and contained per liter 1040 mg Na2HPO4, 3400 mg KH2PO4, 26 mg CaCl2·2H2O, 30 mg MgSO4, 7.5 mg Na2MoO4·2H2O, 0.3mg MnSO4, 8000 mg glucose, 500 mg casein hydrolysate, 36 mg ferric citrate, 10 mg para-aminobenzoic acid, 5 mg biotin, and 1 mg vitamin B1, supplied with 10 mM (NH4)2SO4 (KPM-HN) for pregrowth or 10 mM glutamate (KPM-LN) for nitrogenase activity assays. Antibiotics were supplemented as required at the following concentrations: 100 μg/mL of ampicillin, and 20 μg/mL of gentamycin.

### Quantitative Real-time PCR.

After harvesting bacteria from LB medium, purification of total RNA was performed using RNAiso Plus reagent (TaKaRa, Japan) following the protocol described by the manufacturer. One microgram of qualified total RNA was subjected to reverse transcription with a PrimeScript RT reagent Kit with gDNA Eraser per the manufacturer’s instructions (TaKaRa, Japan). qRT-PCR of the resulting cDNA was performed with gene-specific primers (Table 1) on a CFX Connect Real-Time PCR Detection System (Bio-Rad, USA) with a SYBR Premix Ex Taq (Tli RNaseH Plus) Kit (TaKaRa, Japan). Standard curves of cDNA dilutions were used to determine the PCR efficiency. An expression data analysis was performed by the Pfaffl method of relative quantification using CFX Manager 3.1 software (Bio-Rad, USA).

### Nitrogenase Activity Assay.

The C2H2 reduction method was used to assay nitrogenase activity. EJNC was initially grown overnight in KPM-HN medium and then diluted in 2 mL KPM-LN medium in 20 mL sealed tube to a final OD600 of about 0.3. Air in the tubes was repeatedly evacuated and replaced with argon. After incubation at 37 °C for 6 to 8 h, 2 mL C2H2 was injected. 1 mL of gas was sampled from the gas phase 16 h later and analyzed with a GC-7890B (Agilent, USA) gas chromatograph after appropriate 10-fold serially dilution with nitrogen. Both EJ and EJN severed as controls.

### ICP-MS(Inductively Coupled Plasma Mass Spectrometry) measurement of Cd2+ adsorption.

*Escherichia coli* BL21 containing *OmpA-PbrR*-PJQ200SK (pBAD33) plasmid was cultured in LB medium to an OD600 of 0.4-0.6. Arabinose and CdCl2 were added to the medium to a final arabinose concentration of 40 μM and a final Cd2+ concentration of 100 μM, to induce the formation of CdS nano semiconductors.From the start of the induction, 5 ml of the bacterial solution was taken from the culture every 6 hours (sampling to 24 hours), centrifuged at 4000 rpm for 2 minutes, and washed three times with water to remove the medium involved in the bacterial surface.The washed bacteria were resuspended in 5 ml of water. OD600 was measured, and the bacteria were collected by centrifugation.3 ml of concentrated nitric acid was added and the mixture was digested overnight at 90 °C.The Cd2+ content in the sample was measured using ICP-MS.

### Cd2+ toxicity test.

Multiple groups of LB medium were prepared, and arabinose with a final concentration of 40 μM and different amounts of CdCl2 were added to the medium to form a Cd2+ gradient of 0,150 μM, 300 μM, 600 μM, and 1000 μM.*E. coli* BL21 containing the *OmpA-PbrR*-PJQ200SK (pBAD33) plasmid and plasmid-free *E. coli* BL21 (control) were cultured in different media.The OD600 value was measured every 2 hours and measured for 12 hours.

### Transmission electron microscopy with energy-dispersive x-ray spectroscopy (TEM-EDX).

After the Cd2+ adsorption induction was completed, the bacteria were collected by centrifugation and resuspended in ultrapure water. Samples were sent for TEM image acquisition.The thick carbon film (20 to 30 nm) on the copper grid was immersed in the bacteria solution for 1 second before imaging, dried under atmospheric conditions, and then imaged using TEM. At the same time, the EDX system (EDAX, AMETEK) was attached to the microscope for elemental analysis. All TEM images were imaged using a JEOL JEM-2100 electron microscope at an acceleration bias of 200 kV.

### Characterization of biologically precipitated CdS nanoparticles

The photocatalytic MV2+ reduction assay was performed using a 10-mm quartz cuvette with a cap and a light source(350-W Xe lamp). *E.coli* cells containing biosynthesized CdS nanoparticles were harvested from LB medium by centrifugation (4000 rpm for 10 min). The reaction system consisted of the same amounts of different semiconductors [TiO2 anatase (*10*) and synthesized free CdS nanoparticles (*29*)] and 3ml of 100 mM tris-HCl(PH 7), 150mM NaCl, 5% glycerol, 100mM ascorbic acid, and 5mM MV2+ in the quartz cuvette. O2 was removed by bubbling N2 into the solution for 30 min. The reaction was initiated by light irradiation and stopped by centrifugation and separation of *E.coli*-CdS nanoparticles from the MV buffer. The absorption spectra were immediately measured after centrifugation (1000*g* for 1 min). The amount of reduced MV2+(MV+) that formed was calculated by monitoring the OD605 using the molar conversion coefficient ɛ=1.3 × 104 M-1 cm-1.

|  |  |  |
| --- | --- | --- |
| **Strains** | ***E. coli*** | **Source** |
| EJ | *E. coli* JM109 | TaKaRa |
| EJN | *E. coli* JM109 harboring plasmid pUC57-*nif* | This study |
| EJNC | *E. coli* JM109 harboring plasmids pUC57-*nif and* pJQ200SK-OmpA/PbrR | This study |
| **Plasmids** | **characteristic** | **Source** |
| pUC57 | Cloning vector; pMB1 *ori*; Ampr | Lab store |
| pUC57-*nif* | pUC57 with *nif*; pMB1 *ori*; Ampr | Chemically synthesized |
| pJQ200SK | Cloning vector;p15A *ori*; Gmr | Lab store |
| pJQ200SK-*OmpA/PbrR* | pJQ200SK with *OmpA/PbrR*; p15A *ori*; Gmr | This study |
| **PCR Primers** | **sequence** | **Amplicon** |
| P200F | 5’-GCTCTAGACATGAAAAAGACAGCTATCGCGA | *OmpA/PbrR* |
| P200R | 5’-TCCCCCGGGTCAGATCTTATCGTCGTCATC |
| **qRT-PCR** **Primers** | **sequence** | **Amplicon** |
| QnifBF | 5’-TCGGCCGTGCCAAGGAATTT | *nifB*for  qRT-PCR |
| QnifBR | 5’-CCTATGCCGGACGACAGCAG |
| QnifHF | 5’-TGCGCCGTATGACCGTTACC | *nifH* for  qRT-PCR |
| QnifHR | 5’-CCGGACGCCTCAGCTTTGTT |
| QnifDF | 5’-GCCCGACCAAGACGATGGAG | *nifD* for  qRT-PCR |
| QnifDR | 5’-CCGCAGTCCGCCAATCAGAA |
| QnifKF | 5’-ACCTGAAGTTCGCGGCCAAA | *nifK* for  qRT-PCR |
| QnifKR | 5’-ATCCGGAGCCTGCTCTTCCA |
| QnifEF | 5’-TGCGGCAGATGGCTTACCTG | *nifE* for  qRT-PCR |
| QnifER | 5’-AGCACTGCCCGCTTTCCTTT |
| QnifNF | 5’-TCGAGAGCCGATTGCCGTTC | *nifN* for  qRT-PCR |
| QnifNR | 5’-ATCCAGCGCCTCCTCCAGAT |
| QnifXF | 5’-CGACGGAAGACGGTGTGCAT | *nifX* for  qRT-PCR |
| QnifXR | 5’-TCCAGGAACTGGACGCCTGA |
| QnifVF | 5’-TGGGCGCTGACCATTCGTTT | *nifV* for  qRT-PCR |
| QnifVR | 5’-ACTGCAGCCAGCGCCTTAAA |
| Q16SF | 5’-ACTCCTACGGGAGGCAGCAG | 16S rRNAfor qRT-PCR |
| Q16SR | 5’-ATTACCGCGGCTGCTGG |

## Reference

1. Wang, L., et al., *A minimal nitrogen fixation gene cluster from Paenibacillus sp. WLY78 enables expression of active nitrogenase in Escherichia coli.* PLoS Genet, 2013. **9**(10): p. e1003865.
2. Fixen, K.R., et al., *Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium.* Proc Natl Acad Sci U S A, 2016. **113**(36): p. 10163-7.
3. Brown, K.A., et al., *Light-driven dinitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid.* Science, 2016. **352**(6284): p. 448-50.
4. Kuypers, M.M.M., H.K. Marchant, and B. Kartal, *The microbial nitrogen-cycling network.* Nat Rev Microbiol, 2018. **16**(5): p. 263-276.
5. Wei, W., et al., *A surface-display biohybrid approach to light-driven hydrogen production in air.* Sci Adv, 2018. **4**(2): p. eaap9253.
6. Wang, X., et al., *Using synthetic biology to distinguish and overcome regulatory and functional barriers related to nitrogen fixation.* PLoS One, 2013. **8**(7): p. e68677.
7. Yang, J., et al., *Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity.* Proc Natl Acad Sci U S A, 2017. **114**(12): p. E2460-E2465.
8. Yang, J., et al., *Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology.* Proc Natl Acad Sci U S A, 2018. **115**(36): p. E8509-E8517.
9. Yang, J.G., et al., *Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli.* Proceedings of the National Academy of Sciences of the United States of America, 2014. **111**(35): p. E3718-E3725.
10. Howard, J.B. and D.C. Rees, *Structural basis of biological nitrogen fixation.* Chemical Reviews, 1996. **96**(7): p. 2965-2982.

**Address**

Life Science Department
#163 Xianlin Blvd, Qixia District
Nanjing University
Nanjing, Jiangsu Province
P.R. of China
Zip: 210046

**Email**

nanjing\_china@163.com

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# Team:Nanjing-China/Experiments

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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:Nanjing-China/Results

Nanjing-China2018

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## Overview

This year we aim to achieve light-driven nitrogen fixation based on nitrogenase and OmpA-PbrR. So we divided our experiments into 2 sections: Verification and characterization of CdS semiconductor and Authentication of nitrogenase in *E. coli* cells.

To demonstrate that our design, we set the *E. coli* JM109 strain containing both *nif* gene cluster and *OmpA-PbrR* gene (abbreviated as EJNC in the following passage) as the experimental group. while *E. coli* JM109 (abbreviated as EJ) that doesn’t contain any of those genes was set as a control group.

## Verification and characterization of CdS semiconductor

![](https://static.igem.org/mediawiki/2018/8/8f/T--Nanjing-China--toxicity.jpg)

Figure 1. Cd2+ toxicity test. Cadmium ions shows no significant toxic effects on both strains.

In order to determine the optimum Cd2+ concentration of the system, we conducted a Cd2+ toxicity test. The existence of cadmium ions shows no significant toxic effects on both strains which is evidenced by the virtually equal colony forming units, though a short term growth readjustment brought by heterologous gene expression does occur in EJNC. Considering that, we select 150 μM as the Cd2+ concentration for our follow-up experiments.

![](https://static.igem.org/mediawiki/2018/0/05/T--Nanjing-China--ICP-MS.jpg)

Figure 2. Cd2+ absorption test. The introduction of OmpA-PbrR confers the host cell with Cd2+ absorption capacity.

The amount of biosynthesized CdS semiconductor on the *E. coli* cell surface was measured using inductively coupled plasma mass spectrometry (ICP-MS). It confirms the surface-displayed PbrR-mediated biological precipitation of CdS semiconductor on the outer membranes of cells.

![](https://static.igem.org/mediawiki/2018/a/a7/T--Nanjing-China--TEX-EDX.jpg)

Figure 3. (a) TEM images of biosynthesized CdS nanoparticles on the surface of a EJNC cell.(b) EDX confirmation of randomly chosen CdS nanoparticle. The absorbed Cd2+ precipitates on the outer membrane of EJNC in the form of CdS nanoparticles.

To acquire a fuller understanding of CdS nanoparticles’ characteristics, we performed transmission electron microscopy (TEM) analysis of CdS nanoparticles. The morphology and particle size of CdS nanoparticles are shown in TEM images. Next, we conducted energy-dispersive x-ray spectroscopy (EDX) to analyze its elemental composition. The result demonstrates that the semiconductor on cell surface is mainly composed of cadmium and sulfide.

![](https://static.igem.org/mediawiki/2018/a/a9/T--Nanjing-China--UV.jpg)

Figure 4. Characterization of biologically precipitated CdS nanoparticles on the outer membranes of *E. coli* cells. The UV-Vis Spectrum of *E. coli*/CdS hybrids in solution demonstrating a band gap at 424 nm.

We performed ultraviolet-visible (UV-vis) spectral measurements to directly determine the optical band gap and photocatalytic capability of these CdS semiconductor. The lowest-energy transition of the biosynthesized CdS nanoparticles was detected in the visible region of the solar spectrum (Eg = 2.92 eV, λabsorption = 424 nm), verifying its photocatalytic ability.

![](https://static.igem.org/mediawiki/2018/b/be/T--Nanjing-China--Figure_S4.jpg)

Figure 5. Quantitative comparison of the photoelectrical capacity of in situ biosynthesized CdS nanoparticles. The concentrations of reduced methylviologen (MV) in various experimental groups confirm that the CdS nanoparticles precipitate on the EJNC cells adsorb a photon and transfer an electron to MV2+.

The redox dye methylviologen (MV2+) is a well-established electron mediator. In combination with MV2+, the *E. coli*/CdS hybrids system easily serve as a biocatalyst for photosynthesis. The concentrations of reduced MV in various experimental groups were measured under anaerobic conditions, confirming that CdS semiconductor transfers an electron to MV2+ for every photon it absorbs.

## Authentication of nitrogenase system in *E. coli* cells

![](https://static.igem.org/mediawiki/2018/4/47/T--Nanjing-China--qRT-PCR.png)

Figure 6. Expression profiles of each structure gene in the *nif* cluster that overexpressed in EJNC. Relative expression compared to the housekeeping gene 16S rRNA is shown. qRT-PCR analysis demonstrates that all the component genes of the *nif* cluster are significantly over expressed in EJNC whereas the transcription of these genes are no detected (N.D.) in nondiazotrophic *E. coli* JM109.

To verify the expression of *nif* gene cluster, we conducted Real-time Quantitative PCR(qRT-PCR) to detect the transcriptional level of each *nif* gene in engineered *E. coli*, using 16S DNA as an internal reference. The result provide the relative expression level of each *nif* gene in our constructed *E. coli* strain. After comparing the result with the nif gene cluster expression in *Paenibacillus polymyxa CR1*, we modeled the transcription and planned to optimize the structure of *nif* gene cluster to achieve the best stoichiometric proportion of each *nif* gene. [(see modeling for more details)](https://2018.igem.org/Team%3ANanjing-China/Model)

![](https://static.igem.org/mediawiki/2018/9/90/T--Nanjing-China--aca-1.png)

![](https://static.igem.org/mediawiki/2018/e/ee/T--Nanjing-China--aca-2.png)

Figure 7-13. Acetylene reduction assay.

Nitrogenase can not only reduce dinitrogen to ammonia, but also reduce acetylene to ethylene. Therefore, we performed acetylene reduction assay to indirectly detected its nitrogen fixation activity. First, we plugged in 2ml of engineered bacteria (OD600≈0.3) in a 20ml thermo gas injection bottle, and injected Ar gas into the bottle to remove the air as much as possible. Since the removal of air is definitely not complete, it was necessary to cultivate for 6h to exhaust the oxygen through bacterial respiration. We then took 2ml of acetylene and injected it into a gas collecting bottle after oxygen removal in culture. The culture then reacted in our device for 16h. After that, we took 1ml of the gas from the post-reaction gas collection bottle. In order not to exceed the gas chromatography’ s range, 1ml sample gas was injected into a gas-collecting bag filled with nitrogen to dilute the sample gas. Finally, we used gas chromatography to measure ethylene content in sample. Our sample has been sent for testing by the time Wiki freeze, and we anticipate to receive the result within 5 days. We hope to share our results at the Giant Jamboree.

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Nanjing University
Nanjing, Jiangsu Province
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