Biofilm Parts：
The following steps work for the experiments which conduct the characterization of biofilm via solution.
Part I Preparation for the Characterization: The activation of the bacteria.
Take the CsgA - Histag mutant E.coli as a specific example.
i.First, prepare a aseptic condition with the usage of Bunsen burner, which is able to achieve sterilization in a limited extent.
ii.Take 5ml Luria-Bertani liquid medium into the centrifuge tube with both the LB medium and the centrifuge tube sterilized.
iii.Take 34mg／ml Chloramphenicol which is kept in minus 20 centigrade and diluted 10 times in the 5ml LB liquid medium.
iv. Take the CsgA - Histag mutant E.coli which is kept in minus 80 centigrade into the centrifuge tube with the Chloramphenicol added.
Part II Characterization of Biofilm
i.Take out the CsgA - Histag mutant E.coli which is activated in Part I.
ii. Centrifuge the mutant E.coli with 5000 g for 1minute and leave the supermatant.
iii. Prepare the liquid medium as the following components.
Treat and Control group:
50ml M63 + 0.5ml 20%Glucose + 50ul MgSO4 + 50ul 1mol／L chloramphenicol + 50ul 250ug/L etracycline
Control Group：50ml M63 + 0.5ml 20%Glucose + 50ul MgSO4 + 50ul 1mol／L chloramphenicol
iv.Add the two liquid medium separately to the two CsgA - Histag mutants after centrifugation, and mix them separately into blend.
v.Add 20ml liquid medium specifically to two plates, and add co-responding mutants into them for 200ul target mutants.
vi.Keep the two plates cultivated in temperature 30 degree.）for 48h.
The following steps work for the experiments which conduct the characterization of biofilm via CongoRed Plates.
Part I Preparation for the Characterization: The activation of the bacteria.
Take the CsgA - Histag mutant E.coli as a specific example.
i.First, prepare a aseptic condition with the usage of Bunsen burner, which is able to achieve sterilization in a limited extent.
ii.Take 5ml Luria-Bertani liquid medium into the centrifuge tube with both the LB medium and the centrifuge tube sterilized.
iii.Take 34mg／ml Chloramphenicol which is kept in minus 20 centigrade and diluted 10 times in the 5ml LB liquid medium.
iv. Take the CsgA - Histag mutant E.coli which is kept in minus 80 centigrade into the centrifuge tube with the Chloramphenicol added.
Part II Preparation for the CongoRed Plates.
Make the CongoRed Plates with the components as the following:
i. casamino acids 1g/100ml;
ii.yeast extract 0.1g/100ml;
iii.agar 2g/100ml;
iv. Chloramphenicol 5mg/100ml;
v. ATC 5mg/100ml;
vi. CongoRed 2.5mg/100ml;
vii. Brilliant Blue G250 0.5mg/100ml;
Part III Characterization of biofilm in CongoRed Plates.
Take the activated CsgA-Histag mutant E.coli in small amount into the plates and cultivate them for 48 hours and then the CongoRed plates will show red biofilm vividly if the experiment succeed.
The following steps work for the experiments which can dye the biofilm with the crystal violet.
i.Add 400 ul 0.1% crystal violet dye to the medium, and hatch for 10 to15 minutes.
ii.Wash the medium with ddH2O for 3 to 4 times with the aim of clean the dye;
iii.Use the ChemiDoc MP Imaging System to take pictures of the result;
iv. More steps for quantitively show the results of biofilm:
Take the medium from step ii and add 400ul 30% acetic acid.
Hatch the medium for 10 to 15 minutes, and take the ratio of 1:5ul for the crystal violet-acetic acid solution into new hole plates.
Detect the OD value at 5590nm and make the 30% acetic acid as the control group.
The following steps work for the MCherry-Spytag binding.
i. Adsorb the M63 medium in previous experiments which is used for cultivating biofilm.
ii. Add mixed solution of 4ml PBS, 0.5% Tween, and 1% BSA to the medium.
iii.Add 200ul extracted solution of MCherry-Spytag, and cultivate for 30 hours.
iv. Discard the medium and take off the biofilm from the petri dish, and wash it for 2 times with PBS solution.
v.Use 500ul PBS solution to re-suspend the biofilm.
vi.Take 20ul of the solution in step v to make the sample and observe it with fluorescence microscope.
The following steps work for sample-making for electron microscope.
i. Re-suspend the biofilm got from previous experiments with 200ul PBS solution.
ii. Take 10ul solution from step i and drop it onto the copper net and subside it for 5 to 10 minutes.
iii.Use filter paper to dry the copper net, drop 10ul ddH2O and dry it instantly.
iv.Drop 10ul UAC and stay adsorbed for 20 seconds then dry it with filter paper.
v.Use the infrared lamp to dry it for 15 minutes and get the sample.
The following steps work for the Nanomaterial Templating Test.
i. Add CoCl2 solution to the solution of QDs and NRs which are already finished exchanging of ligands at the ratio of 51to1.
ii. Add nanomaterial to the solution of biofilm we got in the previous experiments as the ratio of 20 to 1.
iii.After cultivating for 1 hour, use PBS solution to wash the medium for 2 times.
Hydrogen Assay Protocol
i. Shake the bacteria overnight for 12 to 16 hours;
ii.Use the volume ratio as 1:50 to inoculate four LB mediums which contain ferric citrate 100mM;
iii.Shake the bacteria for 3 hours and detect the OD value at 600nm
iv.Achieve the state where OD=0.6-0.8, then add a terminal concentration of 1.5mM IPTG
v.100 RPM for an hour at 28 degree.
vi.Keep the sample in the sample bottle sealed overnight for 12 hours.
vii.Set the sample in 4 degree for 20hours.
viii. Hydrogen assay.
QDs subgroup:
Synthesis of CdSe QDs
1. Equipment and Materials
(1) Equipment
Dual vacuum-line, 10ml flasks, 50ml flask, 500ml flasks, 25ml three-necked flask, constant temperature magnetic stirrer, heating block, aluminum foil, house vacuum, vacuum oven, stirrer, droppers, stirring rod, Buchner funnel, Buchner flask, Allihn condenser, rubber tubes, syringes, pipettes, electronic balance, rubber stoppers, centrifuge, ultrasonic cleaning machine.
(2) Materials
Tetramethylammonium hydroxide(TMAH) in methanol solution(25%), selenium powder(99.999%), Cadmium acetate dehydrate(98.5%), stearic acid(90+%), methanol, ethanol, chloroform, acetone, 1-octadecene(ODE, 90%), n-butylamine(98%), n-hexane, dinitrogen.
2. Procedures
(1) Synthesis of CdSt2
Tetramethylammonium hydroxide (TMAH) in methanol solution (20mmol, 7.25ml) and 93ml of methanol mixed in a 500ml flask, stirring 20min, to dissolve stearic acid(HSt, 20mmol, 5.6896g).Cadmium acetate dehydrate(CdAc2·2H2O, 10mmol, 2.6653g) was dissolved in 20ml of methanol in a 50ml flask. Added CdAc2 solution via a dropper into the mixture of TMAH, methanol and HSt drop by drop, stirring rapidly during the addition, to get the white precipitate of CdSt2. Filtered the white precipitate using house vacuum and washed with methanol 3 times, and then dried in the vacuum oven overnight.
(2) Synthesis of CdSe
Weighed out 0.1mmol(0.0678g) of CdSt2 and 4ml of 1-octadecene(ODE, 90%) and mixed in a 25ml three-necked flask, vacuumizing and stirring in N2 atmosphere. Then this reaction system was heated to 250℃. 0.15mmol(0.0188g) of Se powder was added into 3ml of ODE and was dispersed in ODE by using ultrasonic cleaning machine. Injected 1ml of the Se solution into the 25ml three-necked flask quickly. Then cooled the reaction system to 220℃ and stablized at this temperature to allow the growth of CdSe nanocrystals.
(3) Separation of CdSe
1ml of solution of CdSe nanocrystals was added into the mixture of 0.05ml of n-butylamine(98%) and 2ml ethanol in a 10ml flask, heating 5 minutes at 50℃. Discarded the upper alcohol phase after the centrifugation. Then 0.5ml of n-hexane and 2ml ethanol were added, heating 5 minutes at 50℃. As the same, discarded the upper alcohol phase after the centrifugation. Added 0.05ml chloroform, 0.5ml n-hexane and 1.5ml acetone in turn, then centrifugalized to get the precipitate of CdSe quantum dots.
Synthesis of CdS NRs
1. Equipment and Materials
(1) Equipment
Dual vacuum-line, 50ml flasks 100ml flasks, 500ml flasks, 25ml three-necked flask, constant temperature magnetic stirrer, heating block, aluminum foil, stirrer, rubber tubes, syringes, pipettes, electronic balance, rubber stoppers, centrifuge.
(2) Materials
Cadmium oxide(CdO, 99.99%), octadecylphosphonic acid(ODPA,99.99%), trioctylphosphine oxide(TOPO,99%), hexylphosphonic acid(HPA, 99%), hexamethyldisilathiane((TMS)2S, synthesis grade), tributyphophine(TBP, 97%), trioctylphophine(TOP,97%), sulfur(S, 99.998%), methanol, toluene, acetone, ethanol, chloroform.
2. Procedures
(1) Synthesis of CdS seeds
Mixed CdO (0.603), ODPA(0.603g),and TOPO(3.299g) in a 25ml three-necked flask and this reaction system was vacuumized and then heated to 300℃ at N2 atmosphere to allow CdO to dissolve. After CdO dissolved, cooled the solution to 120℃ and degassed it for about 30 minutes. Heated the reaction system and stabilized the temperature at 320℃ at N2 atmosphere. Injected sulfur stock solution(mixture of (TMS)2S(0.179g) and TBP(3g)) quickly to allow the nanocrystals to grow at 250℃ for 7min. Cooled the reaction system quickly to stop the reaction, and then toluene was injected in to get the precipitate of CdS seeds.
(2) Synthesis of CdS nanorods(NRs)
Weighed CdO(0.086g), TOPO(3g), ODPA(0.290g),HPA(0.080g) in a 25ml three-necked flask, vacuumized at 120℃ and heated to 350℃ at N2 atmosphere. TOP(1.5ml) was injected in the mixture after 30 minutes. Stablized the temperature of the solution at 350℃ and then injected the seed-containing solution(0.124g of sulfur in 1.5ml of TOP with 8*10^-8mol CdS seeds) quickly in it. Cooled the solution after 7min to stop the growth of nanorods.
(3) Separation of CdS NRs
Get the precipitate by adding a mixture of acetone, toluene and methanol(1:1:1) and redissolved it by toluene, and then get precipitation by methanol. The precipitate was dissolved in chloroform and precipitated again by ethanol. Using toluene to dissolve the final product.
Synthesis of (1S)-N-[5-[(4-Mercaptobutanoyl)amino]-1-carboxypentyl]iminodiacetic acid(HS-NTA) ligands
1. Equipment and Materials
(1) Equipment
10ml flasks, 100ml flask, 500ml flasks, 250ml three-necked flask, constant temperature magnetic stirrer, house vacuum, vacuum oven, stirrer, droppers, Buchner funnel, Buchner flask, rubber tubes, syringes, pipettes, electronic balance, rubber stoppers.
(2) Materials
Bromoacetic acid, N6-Carbobenzyloxy-L-lysine(Cbz-lys, 98%), NaOH(AR), HCl(GR), Pd/C(10%), methanol(CP), pentane(AR), NaHCO3, 4-butyrothiolactone, acetic acid.
2. Procedures
(1) Synthesis of (1S)-N-(5-Carbobenzyloxyamino-1-carboxypentyl)iminodiacetic acid (Cbz-NTA)
Weighed out 30mmol(8.34g) bromoacetic acid and dissolved it in 30ml of 2M NaOH solution, and then cooled the solution in ice bath. Weighed out 30mmol(8.4g) Cbz-lys and dissolved it in 45ml of 2M NaOH, and added this soulution into the cool solution of bromoacetic acid drop by drop. After stirred at 25℃ overnight, the mixed solution was heated to 70℃, stirring about 2 hours, and cooled to room temperature. Added HCl(1M) into the solution to get the white precipitate. Then NaOH(1M) was added into in order to redissolve the precipitate. Added HCl(1M) again and filtered the final precipitate by house vacuum. Collected the product and dried at 30℃ in the vacuum oven overnight.
(2) Synthesis of (1S)-N-(5-Amino-1-carboxypentyl)iminodiacetic acid(NH2-NTA)
Mixed 15mmol(6g) Cbz-NTA with Pd/C(2g) in a 250ml three-necked flask in a glove box. Injected 100ml methanol into the flask and stirred the solution at H2 atmosphere at room temperature. Filtered the solution and washed with H2O. Removing H2O by using reduced pressure distillation, and then triturated with pentane to get the paste. The paste was dissolved in 20ml H2O and ethanol was added to make the solution cloudy. Heated the cloudy solution to 42℃ to allow it to be clear and then cooled it. Filtered it and got the white crystals. Dried the crystals in the vacuum oven at 30℃ overnight to get NH2-NTA.
(3) Synthesis of HS-NTA
NaHCO3(11.9mmol, 1g) and 4-butyrothiolactone(5.9mmol, 0.6g) were dissolved in 10ml of ddH2O, then NH2-NTA(3.8mmol, 1g) was added to the solution, then stirred the solution overnight at 72℃ and cooled it to room temperature. Regulated pH of the solution to 3 by acetic acid and got the colorless paste, using reduced pressure distillation. The paste was added to ethanol and recrystallized. Filtered the product and washed 3 times with ethanol and pentane respectively to get final product. Then it was dried in a vacuum oven at 30℃ overnight.
Protein extract：
1. Bacterial transformation
2. LB plate screening (four-zone coating)
3. Monoclonal selecting to 50ml centrifuge tube with four kinds of resistants (Kanamycin. Streptomycin. Ampicillin. ) and 15ml LB. Overnight culture.
4. Cultures grown overnight were diluted 1:100 into 25 ml of fresh medium, grown at 37°C , 250rpm until the OD600 reached 0.7-0.8.
5. IPTG (1.5 mM) was added, and the cultures were shaken at 100 rpm for 1 h at room temperature.
6. the cultures were transferred to a sealed blue-lipid flask and fully-filled, using aluminum foil and sealing film to separate O2.16 ℃, overnight culture or room temperature 4h.
7. Cells expressing StrepII- tagged [FeFe] hydrogenase were collected by centrifugation at 16,000 g for 20 min.
8. The cell pellet was resuspended with KPI buffer (3.16g/L KH2PO4 5.16g/L K2HPO4 33.75g/L NaCl ph7.20)
9. Centrifugation at 4,000 g for 10 min. KPI buffer resuspended.
10. Use ultrasound or vacuum or guanidine hydrochloride to break bacteria
Ultrasound breaker:
Preparation of pre-ultrasonic bacteria: After centrifugation of bacteria, the bacteria will be washed with PBS 2-3 times, and then according to the original bacteria liquid volume of 1 / 5-1 / 10 lysis solution re-suspended bacteria, lysate composition : 50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 100 mM NaCl, lysozyme to 100 ug / ml, 0.1% Triton X-100.
Ultrasonic crushing conditions are generally 300w, 10s / 10s, 20 minutes. With Ice bathing.
11. Centrifugation at 4,000 g for 10 min. The solution were blinded with a Nickel affinity column for 1.5h.
12. Centrifugation at 800 g for 5 min. Collecting beads.
13. Use 20,40,300,1000 mM imidazole elution buffer to wash off.
14. Use extract column to centrifugation at 5,000 g for 10 min. Collecting the liquid.
15. SDS-PAGE and Western blotting. For sodium dodecyl sulfate (SDS)-poly- acrylamide gel electrophoresis (PAGE), protein samples were diluted in 4 SDS-PAGE loading buffer, boiled for 10 min, and cooled on ice. Samples were loaded onto a 12% SDS gel and run at 45 mA for 2 h. Following electrophoresis, proteins were blotted onto polyvinylidene difluoride membranes and detected with a StrepTactin-alkaline phosphatase conjugate detection kit (IBA).
