Team:Nanjing-China/notebook

January 2014~March 2014

April 2014

May 2014

June 2014

1th July 2014~7th July 2014

8th July 2014~14th July 2014

14th July 2014~21th July 2014

22th July 2014~31th July 2014

1th August 2014~7th August 2014

8th August 2014~14th August 2014

15th August 2014~21th August 2014

22th August 2014~31th August 2014

1th September 2014~15th September 2014

16th September 2014~30th September 2014

1th October 2014~15th October 2014

1 Bacterial Transformation

Reagents and Materials:
Plasmid DNA (from purified plasmid solution or ligation reaction)
Competent cells (K12 or DH5α)
LB agar plates (containing specific antibiotics)
Liquid LB medium (without antibiotics, sterilized)
	Protocols:
1. Take specific competent cells out of -80℃ refrigerator, and put them on the ice.
2. Put purified Plasmid solution(5μL) or ligation product(10μL) into the competent cell solution , mix it gently and hold it on the
 ice for 30 minutes.
3. Hold the mixture in 42℃ water bath for exact 90 seconds.
4. Put 900μL LB liquid medium in the mixture, put the EP tubes holding mixture in to the shaker, and shake for 1 hour at 37℃ at 
proper speed.
5. Centrifuge the solution at 3000rpm for 90s.
6. Remove 860 μL solution, blend the remainder, put it on the LB plates( containing specific antibiotics and having been 
activated for more than 20 minutes ) and coat the plates with sterile glass spreading rod.
7. Cultivate the bacterial for more than 10 hours.

Attention: 2,4,6 are all done in super clean bench, which should be sterilized by ultraviolet for more than 15 minutes before use.


2 Colony PCR
	Reagents and Materials:
		rTag DNA polymerase( in glycerol, TaKaRa taq)
10X PCR buffer(Mg2+ free, TaKaRa)
25mM MgCl2 solution(TaKaRa)
dNTP mixture(TaKaRa)
ddH2O
	Protocols:
		Mixture(per reaction)
			28μLddH2O
			5μL 10X buffer
			3μL MgCl2
			1μL dNTP
			1μL primer(F)
			1μL primer(R)
			10μL template
			1μL rTaq
1. Prepare 4 EP tubes and 4 PCR tubes for each plate, put 10μL ddH2O in each EP tubes and 8μL ddH2O in each PCR tubes.
2. Pick up a little bit of colony and mix it with water in EP tubes, put 2μLof the mixture to the corresponding PCR tube with 8μL 
and mix it.
3. Repeat 2 for another 3 colonies in the same plate.
4. Prepare the PCR mixture and place the tubes in the thermal cycler.
5. Set the PCR program in the thermal cycler.
	Stage 1(1 cycle): 95℃for 10 minutes
	Stage 2: 95℃ for 30 seconds, 60℃ for at least 30 seconds, 72℃ for at least 1 minutes. ( for 30 cycles. The time for 60℃ is 
always half the time for 72℃, and the time for 72℃ is 1 minutes/kb )
  	Stage 3: 72℃ for 10 minutes
 	Stage 4: hold at 12℃


3 Bacterial Culture
	(1) Culture in liquid LB medium
Reagents and Materials:
			Liquid LB medium ( with specific antibiotics, sterilized )
Specific antibiotics (A: Ampicillin, C: Chloramphenicol, K: Kanamycin, T: Tetracycline)
		Protocols:
			1. Put 10ml Liquid LB medium in 50ml tubes, and add the antibiotics(antibiotics : Liquid medium = 1 : 1000)
2. Add the bacterial solution to the medium( such as the mixture of bacterial and water after picking up the colony)
3. Put the tubes into the shaker, and shake for more than 10-12 hours at 37℃ at proper speed.
	(2)	Culture on the plate
		Reagents and Materials:
			Plate ( with specific antibiotics)
		Protocols:
1. Put 100-200 μL bacterial solution on the LB plates (containing specific antibiotics and having been activated for more than 20
 minutes) and coat the plates with sterile glass spreading rod.
2. Cultivate the bacterial for more than 10 hours.


4 Bacterial Stock
Reagents and Materials:
	Glycerol (50%)
	Protocols:
1. Put 240μL glycerol in screw cap tubes, and add 760μL overnight liquid medium.
		2. Place screw cap tubes in -80℃ refrigerator.


5 Plasmid Preparation
	Reagents and Materials( AxyPrep Plasmid MiniPrep Kit):
		Buffer S1(with RNase A, stored at 4℃)
		Buffer S2
		Buffer S3
		Buffer W1
		Buffer W2(with ethanol)
		ddH2O
	Protocols:
1. Put 1-4 ml overnight LB liquid medium into 2ml EP tubes, centrifuge tubes at 12000g for 1 minute and discard the
 supernatant.
2. Add 250μL Buffer S1, suspend the bacterial completely.
3. Add 250μL Buffer S2, gently overturn the tubes for 4-6 times. ( This step last less than 5 minutes)
4. Add Buffer S3, gently overturn the tubes for 6-8 times, centrifuge the tubes at 12000g for 10 minutes.
5. Transfer supernatant from step 4 to Miniprep column, centrifuge the tubes at 12000g for 1 minute, discard the filtrate.
6. Place the columns back to 2ml tubes, add 500μL Buffer W1, centrifuge tubes at 12000g for 1 minute, discard the filtrate.
7. Place the columns back to 2ml tubes, add 700μL Buffer W2, centrifuge tubes at 12000g for 1 minute, discard the filtrate.
8. Repeat step 8.
9. Place columns back to tubes, centrifuge tubes at 12000g for 1 minutes.
10. Place columns to 1.5ml tubes, add 60μL 65℃ ddH2O, leave them standing at room temperature for 1 minute, and centrifuge
 them at 12000g for 1 minute.


6 Restriction Digestion
Reagents and Materials:
Specific TaKaRa restriction endonuclease（S: SpeI, P: PstI, E: EcoRI, X: XbaI, N Nde I）
10X Specific TaKaRa buffer( When you use X and P, you should use buffer M. When you use S and P you should use buffer H.
 For other situations, you can refer to the table of buffer for double digestion)
	Protocols:
		1. Prepare the reaction mixture in PCR tubes as follows.
		Plasmid digestion mixture:
			43μL vector
			5μL specific buffer
			1μL enzyme 1
			1μL enzyme 2
		PCR product digestion mixture:
16μL PCR product( after purification )
			2μL specific buffer
			1μL enzyme 1
			1μL enzyme 2
2. Place the tubes in water bath at 37℃ for specific time( The time depend on kinds of the plasmid and activity of the enzyme)


7 Ligation
	Reagents and Materials:
		T4 DNA ligase (TaKaRa)
		10X T4 ligase buffer (TaKaRa)
	Protocols:
		1. Prepare the reaction mixture in tubes.
		Mixture：
			2μL vector
			6μL insert
			1μL T4 ligase buffer
			1μL T4 ligase
       ( The volume of vector and insert depend on the concentration of them, it is better to let the mole ratio of vector: insert to be
 1:10. Total volume of vector and insert should be 8μL)
2. Place the tubes in Low-temperature thermostat bath at 16℃ for more than 2 hours.


8 Agarose Electrophoresis
Reagents and Materials:
	1X TAE buffer
	REGULAR AGAROSE G-10(BIOWEST)
	DL2000 DNA Marker
	10X Loading Buffer
	Protocols:
		1. Prepare the agarose gel (1%).
		Mixture:
			0.5 g REGULAR AGAROSE G-10
			50ml TAE buffer
Heat the mixture until boiling (about 1 minute in microwave oven). Add 5μl EB( ethidium bromide) when it is not very hot, pour it
 into assembled gel pouring apparatus, wait until the gel turn solid enough.
2. Place the gel into the electrophoresis tank, and pour 1X TAE buffer until the gel is covered.
3. Add DL2000 DNA Marker and samples(with loading buffer). Run the apparatus at proper voltage (usually 120V- 135V) for
 proper time (20-30 minutes).
4. Visualize the gel under ultraviolet, and record the results.
	Reagents and Materials:
	Protocols:


9 Plasmid PCR
Reagents and Materials:
rTag DNA polymerase( in glycerol, TaKaRa Taq ) or TaKaRa La Taq
PCR buffer or 10X La Taq RCR buffer ( TaKaRa)
25mM MgCl2 solution (TaKaRa)
dNTP mixture (TaKaRa)
ddH2O
	Protocols:
		Mixture:
			37μL ddH2O
			5μL 10X buffer
			3μL MgCl2
			1μL dNTP
			1μL primer(F)
			1μL primer(R)
			1μL template
			1μL rTaq Polymerase
		1. Prepare the mixture in PCR tubes.
		2. Place the PCR tubes in the thermal cycler, and set the program.
	Stage 1(1 cycle): 95℃for 3 minutes
	Stage 2: 95℃ for 30 seconds, 60℃ for at least 30 seconds, 72℃ for at least 1 minutes. ( for 30 cycles. The time for 60℃ is
 always half the time for 72℃, and the time for 72℃ is 1 minutes/kb )
  	Stage 3: 72℃ for 10 minutes
 	Stage 4: hold at 12℃


10 LB Agar Plate and LB culture medium
Reagents and Materials:
	Tryptone(OXOID)
	Yeast Extract(OXOID)
	NaCl(Sinopharm Chemical Reagent Co..Lid)
	Agar Powder(Solarbio)
	Specific antibiotics
	Protocols(for 1L LB medium):
	  (1) LB liquid cultivation
		Mixture:
			10g Trptone
			5g Yeast Extract
			10g NaCl
		1. Add water to the Mixture until the total volume is 1 L in beaker.
		2. Place the beaker on the magnetic stirrers, and stir it until solid resolves.
3. Sterilize the medium in high-handed sterilization pan at program P7. (the container should be opened partly)
	  (2) Plate
		Mixture:
			10g Trptone
			5g Yeast Extract
			10g NaCl
		1. Add water to the Mixture until the total volume is 1 L in beaker.
2. Place the beaker on the magnetic stirrers, and stir it until solid resolves. Pour the solution into a bottle containing 15g Agar
 Powder.
3. Sterilize the medium in high-handed sterilization pan at program P7. (the container should be opened partly)
4. Add specific antibiotics when it is not very hot and shake it.
5. Pour the LB medium to the plate (25ml/each) , wait until it solidify (done in the super clean bench) and place plates to 4℃
 refrigerator.


11 Primer dissolution
Reagents and Materials:
	ddH2O
	Protocols:
		1. Shortly centrifuge the EP tubes containing primer powder.
		2. Slowly open the tubes, add ddH2O until the concentrate of primer is 20 mM,  vortex them, put the tubes in -30℃
 refrigerator.


12 Making competent cells
Reagents and Materials:
	CaCl2 and glycerol mixture(0.1mol/L CaCl2)， CaCl2-MgCl2(80mmol/LMgCl2, 20mmol/L CaCl2)
   K12 or DH5α plate
	Protocols:
1. Pick a colony from the plate and put it to 100 mL LB medium, and cultivate the medium.
2. Measure OD600, until OD600 is 0.35.
3. Precool two 50mL tubes, 80 EP tubes, CaCl2 and glycerol mixture, CaCl2-MgCl2

4. Put 100ml bacterial solution to two 50ml tubes, and place them on ice for 10 minutes.
5. Centrifuge tubes at 4100rpm for 10 minutes, at 4℃.
6. Discard the supernate. Put the tubes upside down without caps for 1 minute to get rid of the supernate remained.
7. Resuspend the bacterial by 30ml cold 0.1mol/L CaCl2-MgCl2 per 50ml medium at the beginning.
8. Centrifuge at 4100rpm, 4℃ for 10 minutes
9. Discard the supernate. Put the tubes upside down without caps for 1 minute to get rid of the supernate remained.
10. Resuspend the bacterial by 2ml cold 0.1mol/L CaCl2 per 50ml medium at the beginning.
11. Place 50μL mixture in each tube, and place them in -80℃ refrigerator.


13 Gel Extraction
	Reagents and Materials:
		Buffer DE-A
		Buffer DE-B
		Buffer W1
		Buffer W2(with alcohol)
		Isopropanol
		ddH2O
	Protocols:
1. Cut wanted bands on the gel place them in the tubes and calculate their volumes.
2. Pour triple volume of Buffer DE-A in the tubes, keep tubes at 75℃ until the gel melt completely.
3. Add 1.5 times volume of Buffer DE-B, blend it.( If the fragment you want is shorter than 400bp, one volume of isopropanol
 need to be added)
4. Add mixture from step 3 to the columns, centrifuge them at 12000g for 1 minute and discard the filtrate.
5. Add 500μL Buffer W1, centrifuge them at 12000g for 30 seconds and discard the filtrate.
6. Add 700μL Buffer W2, centrifuge them at 12000g for 30 seconds, and discard the filtrate.
7. Repeat step 6.
8. Centrifuge the tubes at 12000g for 1 minute.
9. Place columns to 1.5ml tubes, add 60μL 65℃ ddH2O, leave them standing at room temperature for 1 minute, and centrifuge
 them at 12000g for 1 minute.


14 Purification of PCR product and Restriction Digestion product
Reagents and Materials:
Buffer PCR-A
Buffer W2
ddH2O
	Protocols:
1. Add three volume of Buffer PCR-A to the mixture (if Buffer PCR-A needed less than 100μL, add 100μL ), centrifuge them at
 12000g for 1 minute and discard filtrate.
2. Add 700μL Buffer W2, centrifuge at 12000g for 1 minute, and discard the filtrate.
3. Add 400μL Buffer W2, centrifuge at 12000g for 1 minute, and discard the filtrate.
4. Place columns to 1.5ml tubes, add 60μL 65℃ ddH2O, leave them standing at room temperature for 1 minute, and centrifuge
 them for 1 minute.


    

15 Process with AP enzyme
		Regents and Materials:
			AP enzyme ( Alkaline Phasthatase)
			Buffer( 10XBS buffer W : ddH2O = 6 : 3)
		Protocols:
			Mixture:
				50μL digested product
				9μL Buffer
				1μL AP enzyme
1.blend the mixture, and put it in Low-temperature thermostat bath at 25℃ for 30 minutes.

        Here we express our gratitude to technical support from Prof. Zhao Jing’s lab, Nanjing University.
        We express our gratitude to the great support from Professor Kong Lingdong and Professor Yang Sihai, and the help from
 other teachers from the School of Life Sciences, Nanjing University.
        And especially, we express our heartfelt thanks to:
        2014-Tsinghua-A, for their help in our model building.
        2014-Jilin-China, for their help with the mrlA gene synthesis.
        2014-NCTU_Formosa, for their great seminar.
        Wang Jiangning, for his help in buliding the wiki.
        Finally, we express great thanks to 2014-NJU-QIBEBT and 2013-Nanjing-China for providing us with some important substance,
    

How to make use of our bacteria?

    To solve this problem, we constructed a small device like this:

a normal injector with a filtering membrane front. We will culture our bacteria on the filtering membrane anteriorly. When
used, you just need to take in some dirty water and eject them, then you will get clean water.

Moreover, we try to design a device to clean the water discharge from factories.

As is shown in the picture, if we fix the channel 1 in the end of factories’ tube, after a while, open the channel 3, clean water
will pour out.

How to avoid engineered bacteria hazard and gene transfer?

     Our plasmid consists more than 40 artificial fragments. To avoid hazard caused by engineered bacteria, we construct a suicide system. Bacteria with the "suicide system" will perform like this:

In our lab, we will use IPTG to culture bacteria, and in the nature, bacteria will die without IPTG or Toxin.We proved that it works in our wet lab experiments.We also design some systems to stop horizontal gene transfer. Detail can be seen in our safety section.

How can we popularize iGEM and make people from deferent fields know synthetic biology?

     We establish iGEM club and have finished the new recruit. In the new recruit we attract many students from deferent departments who are interested in synthetic biology. In addition, we are planning to establish an information platform that students from all departments of Nanjing University can share the information related to iGEM. These activities are all supported by teachers of our university. Here are some pictures in the new recruit.

In addition, we also apply for a reward about environment that our university hold.