Difference between revisions of "Team:ShanghaitechChina/Notebook"

Revision as of 18:01, 18 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.

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.

@@ Line 97: / Line 97: @@
 <h2>QDs subgroup:</h2>
 <h3>Synthesis of CdSe QDs </h3>
+We synthesized CdSe QDs using a protocol described in.<p></p>
 <h4><b>1. Equipment and Materials</b></h4>
 (1) Equipment<p></p>
-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.<p></p>
+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, droppers, stirring rod, Buchner funnel, Buchner flask, rubber tubes, syringes, pipettes, electronic balance, rubber stoppers, centrifuge, ultrasonic cleaning machine.<p></p>
 (2) Materials<p></p>
-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.<p></p>
+Tetramethylammonium hydroxide anhydrate(TMAH), selenium powder(99.999%), Cadmium acetate dehydrate(98.5%), stearic acid(90+%), methanol, ethanol, dichloromethane, acetone, 1-octadecene(ODE, 90%), n-butylamine(98%), n-hexane, nitrogen. <p></p>
 <h4><b>2. Procedures</b></h4>
-(1) Synthesis of CdSt2<p></p>
+(1) Synthesis of CdSt2 <p></p>
-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.<p></p>
+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.<p></p>
 (2) Synthesis of CdSe<p></p>
-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.<p></p>
+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.  <p></p>
-(3) Separation of CdSe<p></p>
+(3)Purification and isolation of CdSe<p></p>
-ml 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.<p></p>
+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. <p></p>
 <h3>Synthesis of CdS NRs </h3>
 <h4><b>1. Equipment and Materials</b></h4>
 (1) Equipment<p></p>
-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.<p></p>
+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. <p></p>
 (2) Materials<p></p>
-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.<p></p>
+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. <p></p>
 <h4><b>2. Procedures</b></h4>
 (1) Synthesis of CdS seeds<p></p>
-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.<p></p>
+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.<p></p>
 (2) Synthesis of CdS nanorods(NRs)<p></p>
-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.<p></p>
+.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. <p></p>
 (3) Separation of CdS NRs<p></p>
 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.<p></p>
-<h3>Synthesis of (1S)-N-[5-[(4-Mercaptobutanoyl)amino]-1-carboxypentyl]iminodiacetic acid(HS-NTA) ligands </h3>
-<h4><b>1. Equipment and Materials</b></h4>
-(1) Equipment<p></p>
-ml 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.<p></p>
-(2) Materials<p></p>
-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.<p></p>
-<h4><b>2. Procedures</b></h4>
-(1) Synthesis of (1S)-N-(5-Carbobenzyloxyamino-1-carboxypentyl)iminodiacetic acid (Cbz-NTA)<p></p>
-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.<p></p>
-(2) Synthesis of (1S)-N-(5-Amino-1-carboxypentyl)iminodiacetic acid(NH2-NTA)<p></p>
-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.<p></p>
-(3) Synthesis of HS-NTA<p></p>
-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.<p></p>
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Difference between revisions of "Team:ShanghaitechChina/Notebook"

Revision as of 18:01, 18 October 2016

Biofilm Parts：

Part I Preparation for the Characterization: The activation of the bacteria.

Part II Characterization of Biofilm

Part I Preparation for the Characterization: The activation of the bacteria.

Part II Preparation for the CongoRed Plates.

Part III Characterization of biofilm in CongoRed Plates.

Hydrogen Assay Protocol

QDs subgroup:

Synthesis of CdSe QDs

1. Equipment and Materials

2. Procedures

Synthesis of CdS NRs

1. Equipment and Materials

2. Procedures

Protein extract：