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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 coliJM109 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 (1000g 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	nifBfor  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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