iv.Add the two liquid medium separately to the two CsgA - Histag mutants after centrifugation, and mix them separately into blend. <p></p>
iv.Add the two liquid medium separately to the two CsgA - Histag mutants after centrifugation, and mix them separately into blend. <p></p>
v.Add 20ml liquid medium specifically to two plates, and add co-responding mutants into them for 200ul target mutants.<p></p>
v.Add 20ml liquid medium specifically to two plates, and add co-responding mutants into them for 200ul target mutants.<p></p>
Revision as of 18:10, 19 October 2016
igem2016:ShanghaiTech
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.
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
We synthesized CdSe QDs using a protocol described in.
(1) Synthesis of CdSt2
Tetramethylammonium hydroxide(TMAH,20mmol,1.8230g), stearic acid(HSt, 20mmol, 5.6896g) and methanol(100ml) were mixed in a 500ml flask, stir for 20min to dissolve. Cadmium acetate dehydrate(CdAc2·2H2O, 10mmol, 2.6653g) was dissloved in methanol(20mL) in a 50ml flask. CdAc2 solution was then added into the HSt solution drop by drop and stirred rapidly during the addition to get the white precipitate of CdSt2. The white precipitate was filtered using a suction apparatus, washed with methanol for 3 times, and dried overnight in the vacuum oven.
(2) Synthesis of CdSe
CdSt2(10mmol,0.0678g) and 1-octadecene(ODE, 90%, 4mL) were mixed in a 25ml three-necked flask, degassed and stirred in N2 atmosphere. Then this reaction system was heated to 250℃. Se powder(15mmol,0.0118g) was added into ODE(3mL) and was dispersed in ODE using sonication. To the system, 1ml of the Se solution was injected quickly. The reaction system was cooled to 220℃ and stabilized at this temperature for 3min to allow the growth of CdSe nanocrystals.
(3)Purification and isolation of CdSe
The mixture of 0.05ml of n-butylamine(98%) and 2ml ethanol was added into 1ml of product solution in a 10ml flask, heated for 5 minutes at 50℃. Discarded the upper alcohol phase after the centrifugation. Excessive ethanol was added to the lower phase and then centrifugalized to get the precipitate of CdSe quantum dots. CdSe QDs are then dissolved in CH2Cl2 to preserve.
(1) Synthesis of CdS seeds
Cadmium oxide(CdO, 0.100g), octadecylphosphonic acid (ODPA, 0.603g) and trioctylphosphine oxide (TOPO, 3.299g) were mixed in a 25ml three-necked flask. The reaction system was degassed and then heated to 300℃ under N2 atmosphere to allow CdO to dissolve. After CdO dissolved, the solution was cooled to 120℃ and degassed for about 30 minutes. The system was then heated and stabilized at 320℃ under N2 atmosphere. Hexamethyldisilathiane (TMS)2S(0.179g) and TBP(3g) was mixed to make a sulfur stock solution and then quickly injected to the system. The nanocrystals were allowed to grow at 250℃ for 7.5min. Then the toluene was injected and the reaction system was quickly cooled to room temperature. The product was precipitated with excessive methanol and washed twice with a 1:1 mixture of toluene and methanol. The final precipitate was dissolved in 5mL of TOP.
(2) Synthesis of CdS nanorods(NRs)
0.124g of sulfur in 1.5ml of TOP was prepared beforehand by heating and stirring and at 80℃ after degassing for 20min. After the sulfur dissolved and the system cooled to room temperature, the product solution in (1) with 8*10^-8mol CdS seeds was added to the solution to make a seed containing solution. CdO(0.086g), TOPO(3g), ODPA(0.290g),HPA(0.080g) was mixed in a 25ml three-necked flask, degassed at 120℃ and heated to 350℃ for 30min under N2 atmosphere. After which TOP(1.5ml) was injected in the mixture. The temperature of the solution was stabilized at 350℃ and then to the system the seed-containing solution was quickly injected. The solution was cooled after 7min to stop the growth of nanorods. The product was precipitated with a 1:1:1 mixture of acetone, toluene and methanol and washed twice with methanol. The final product was redissolved with toluene.
(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.
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).
