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

Protocol

Medium

Nitrogen-deficient medium contained (per liter) 10.4 g Na2HPO4, 3.4 g KH2PO4, 26 mg CaCl2N 2H2O, 30 mg MgSO4, 0.3 mg MnSO4, 36 mg Ferric citrate, 7.6 mg Na2MoO4·2H2O, 10 mg p-aminobenzoic acid, 5 mg biotin, 4 g glucose as carbon source and 2 mM glutamate as nitrogen source. Nitrogen-free medium don’t contain glutamate.

Constraction of plasmid

Enzyme digestion

BamH1	1ul
Kpn1	1ul
10*Buffer	3ul
plasmid	20ul
ddH2O	3ul

Total 30ul, react for at least 5 hours.

DNA ligation

T4 DNA ligase	1ul
Ligase buffer	2ul
Inset	14ul
vector	2ul

Total 20ul

 

PCR（pbrr）:

Reactoin system(total 50 ul)

Primer R	1ul
Primer F	1ul
plasmid	1ul
dNTP	1ul
rTap	1ul
10*Buffer	5ul
ddH2O	40ul

Reaction time

95℃	3min
95℃	30s
65℃	30s
72℃	1min	34 cycle from step2
72℃	5min

Constract lighr-driven nitrogen fixion system

Transfer the two plasmid: ompa-pbrr/PBAD and nif gene/PUC57 to the E.coli BL21. The constracted E.coli was cultured in LB medium containing 100ug/ml ampicillin at 37℃. When the optical density at 600nm(OD600) reached 0.6, cultures were transferred to a nitrogen-deficient medium (supplemented with Ampicillin) with 100% N2. During anaerobic growth, cultures were supplemented with a sterile solution contain 1mM IPTG, 1mM Cd(NO3)2, 1mM Arabinose in a nitrogen-deficient medium (supplemented with Ampicillin). Cultures were incubated at 37℃ under anaerobic conditions for 12 hours with stirring and then in light for 3 hours.

Verification of Pnif in E. coli

Measure the effect of ammonium

To verify the promoter of nifB gene(Pnif) in E. coli, a fluorescent assay was conducted. Plasmids with Pnif and Dronpa which encode fluorescent protein were transferred into E. coli. Strains were grown overnight in 100ml LB broth. The strain was then centrifuged and resuspended with 1ml sterilized water. 100μL bacteria solution was transferred to 500mL nitrogen-deficient medium (supplemented with Ampicillin) in a serum vial. For measuring the effect of ammonium, a gradient of 0, 1mM, 5mM and 10mM was built. After incubating the cultures for 15 minutes, the headspace of serum vials was then evacuated and replaced with argon. 1.5ml of the culture was centrifuged and resuspended, and then use a plate reader (Tecan Infinite M1000 Pro) for OD600 and fluorescent (λ_excitation/λ_emission = 503 nm/518 nm) assay every 1 hour. All treatments were in three replicates and all the experiments were repeated three or more times.

Activity curve

To determine the best timing for Pnif  expression, strains mentioned above were grown in 100ml LB broth at day，OD600 and fluorescent assays were conducted by plate reader(Tecan Infinite M1000 Pro) every one hour until the fluorescent/OD600 value stay unchanged. 50mL of the culture was then centrifuged, resuspended with 50ml sterilized water and transferred to 50mL nitrogen-deficient medium (supplemented with Ampicillin) in a serum vial. The headspace of serum vials was then evacuated and replaced with argon. Every one hour, 1.5ml of the culture was centrifuged and resuspended, and then use a plate reader (Tecan Infinite M1000 Pro) for OD600 as well as fluorescent (λ_excitation/λ_emission = 503 nm/518 nm) assays. All treatments were in three replicates and all the experiments were repeated three or more times.
As control Group， the E.coli only with dro gene was used to compare with our recombinant E. coli strains

Demostrate the expression of nitrogenase gene and ompa-pbrr gene

SDS analysis

Cultures of engineered E. coli strains were grown either in non-N2-fixing conditions (21% O₂, dark) and harvested after 15 h of incubation or grown in N2-fixing conditions (100%N₂, light) and harvested after 15 h of incubation(4h in light condition), respectively.
The cell pellet collected from 4 ml cultures at OD600 = 1 was dissolved in 200 ml sodium dodecyl sulfate (SDS) gel-loading buffer, boiled for 5 min and then 20 ul was loaded onto the stacking gel. Proteins were separated and stacked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with 12% separation gel and 5% stacked gel, both with an acrylamide:bis-acrylamide ratio of 29:1.
E. coli containing only the Ompa-Pbrr gene or only the nif gene are set as control groups.

Demostrate the activity of recombinant E. coli

Acetylene Reduction Assay

For nitrogenase activity assays, Recombinant E. coli strains were initially grown in LB medium (supplemented with Ampicillin) for 16h. The cultures were collected by centrifugation, washed three times with sterilized water and then resuspended in nitrogen-deficient medium (supplemented with Ampicillin) to a final OD600 of 0.6-1.0. Then, 50 ml of the culture was transferred to a 150-ml test tube and the test tube was sealed with robber stopper. The headspace in the tube was then evacuated and replaced with argon gas. After incubating the cultures for 6–8 h at 37°C with shaking at 180 rpm, Acetylene (10% of the headspace volume,about 10ml) was injected into the test tubes. After incubating the cultures under illumination for a further 16h, 100 ml of culture headspace was withdrawn through the rubber stopper with a gas tight syringe and manually injected into a HP6890 gas chromatograph to quantify Acetylene reduction.
All treatments were in three replicates and all the experiments were repeated three or more times.
For measuring the effect of oxygen on nitrogenase activity, nitrogen-deficient medium was used, and oxygen was adjusted to the initial concentration indicated at the start of the incubation.
As control Group， the E.coli only with ompa-pbrr gene was used to compare with our recombinant E. coli strains.

Demostrate the activity of recombinant E. coli with light

Colorimetric assay of NH3 production

The amount of NH3 produced was measured using a colorimetric ammonia assay kit (BioVision). Briefly, 50 μL of E.coli（diluting to 1/5 by assay buffer） was mixed with 50 μL of kit reaction buffer and incubated at 37 °C for 1 h. The absorbance at 570 nm was measured by plate reader (Tecan Infinite M1000 Pro).

In order to study the effect of Ompa-Pbrr in transferring electron transport energy under illumination, after 12 h, it was shaken for 3 h in the dark and is set as a control group. E. coli containing only the Ompa-Pbrr gene or only the nif gene are also set as control groups.

Fluorescence assay of NH3 production

Ammonia production was verified by a second, independent method of ammonia detection based on fluorescence detection using o-phthaladehyde. Culture was added to 1 mL of a solution of 20 mM o-phthalaldehyde, 0.2 M phosphate buffer (pH 7.3), 5% ethanol, 3.4 mM β-mercaptoethanol. Samples were incubated in the dark for 30 min at room temperature. The fluorescence (λexcitation/λemission = 410 nm/472 nm) of the solutions was measured using a plate reader (Tecan Infinite M1000 Pro).
A calibration curve was created by incubating E.coli in the dark for 90 min. Ammonium chloride was then added, in appropriate amounts, to aliquots of the filtered solution to a final volume of 50 μL then reacted, incubated, and assayed as described above. Ammonia production above background levels was in agreement with the results of the colorimetric assay.
In order to study the effect of Ompa-Pbrr in transferring electron transport energy under illumination, after 12 h, it was shaken for 3 h in the dark and is set as a control group. E. coli containing only the Ompa-Pbrr gene or only the nif gene are also set as control groups.

Reference

1. Kathryn RF,Yanning Z,et.al.(2016)Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium PNAS
2. Wang L,Zhang L,Liu Z,Zhao D,Liu X et.al(2013) A minimal Nitrogen Expression of Active Nitrogenase in Escherichia coli PLOS Genetics9(10):e1003865
3. Katherine AB,Derek FH,Molly BW et.al(2016) Light-driven nitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid Science352,448
4. Wei W,Sun PQ,Li Z,Song KS,Su WY,Wang B,Liu YZ,Zhao J et.al (2018) A surface display biohybrid approach to light-driven hydrogen production in air Science eaap9253
5. Wei W,Zhu T,Wang Y et.al(2012) Engineering a gold-specific regulon for cell-based visual detection and recovery of gold Chem.Sci,3,1780-1784
6. James BH&Douglas CR(1996) Structural Basis of Biological Nitrogen Fixation Chem.Rev.96,2965-2982