Difference between revisions of "Team:Nanjing-China/Notebook"

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.

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.