Team:Nanjing-China/protocol

From 2013.igem.org

Extraction of total RNA: (1) Pick a single colony from a selective plate and inoculate a culture of 5mL LB medium and incubate until OD600 reaches 0.6 at 37℃ with vigorous shaking.
(2) Harvest the bacteria cells by centrifuge at 12000rpm in 1.5mL microcentrifuge and lyse them by repeatedly inhaling and emitting with a pipette in Buffer RL.
(3) Add 0.2mL chloroform every 1mL RL. Shake violently for 15sec and incubate at room temperature for 3min.
(4) Centrifuge at 12000rpm at 4℃ for 10min. The sample will separate into three layers, the bottom layer is the organic layer, the middle and the top layer are colorless water layers, and RNA persists in the water layers. (The volume of the water layers is approximately 60% of the added RL.) Apply the water layers to a new tube.
(5) Add a volume of 70% ethanol (check if anhydrous ethanol is added) to a volume of the solution from last step and mix thoroughly by inverting. There is possibly some precipitation after this step. Apply the solution the possible precipitation to the column RA.
(7) Centrifuge at 10000 rpm for 45s and discard the flow-through.
(8) Add 500μL Buffer RE. Centrifuge at 12000rpm for 60s and discard the flow-through.
(9) Add 700μL Buffer RW (check if anhydrous ethanol is added). Centrifuge at 12000rpm for 60s and discard the flow-through.
(10) Add 500μL Buffer RW. Centrifuge at 12000rpm for 60s and discard the flow-through.
(11) Centrifuge at 12000rpm for 2min and discard the flow-through.
(12) Place the column in a new clean 1.5mL microcentrifuge tube and add 50-80μL ddH2O (better preheated at 65℃) to the center of the column. Stand at room temperature for 2min. Centrifuge at 12000rpm for 1min.
(13) Apply the flow-through from the last step to the center of the column and centrifuge at 12000rpm for 1min.

(1) Prepare the following mixture in a microtube:

Random 6 mers (50μM) 			1

dNTP Mixture (10 mM each) 		1

Template RNA 				    total RNA less than 5μg

RNase free dH2O	 			 up to 10μL

(2) Keep for 5min at 65℃ and cool immediately on ice.
(3) Prepare the reaction mixture by combining the following reagents to a total volume of 20μL:

Template RNA and Primer Mixture 	10μL

5× PrimeScript II Buffer 			4μL

RNase Inhibitor (40U/μL) 			0.5μl

PrimeScript II RTase( 200U/μl) 		1μL

RNase free dH2O 				up to 20μL

(4) Mix gently.
(5) Incubate the reaction mixture immediately under the following conditions
42℃ 60min (6) Inactivate the enzymes by incubation at 95℃ for 5min, followed by cooling on ice.

PCR

3-1 Colony PCR

(1) System:	2×PrimeStar	12.5μL
	Forward primer	1~2.5μL
	Reverse primer	1~2.5μL
	Bacteria solution	1~2μL
	dd water	up to 25μLL

(2) Steps:	Step 1	94℃
	Step 2	94℃
	Step 3	Annealing temperature
	Step 4	72℃
	Step 5	go to Step 2, 25~35 cycles
	Step 6	72℃
	Step 4	Pause at 4℃

3-2 PCR with DNA as Template

(1) System:	2×PrimeStar	12.5μL
	Forward primer	1μL
	Reverse primer	1μL
	Template DNA	0.5μL
	dd water	up to 25μL

(2) Steps:	Step 1	94℃
	Step 2	94℃
	Step 3	Annealing temperature
	Step 4	72℃
	Step 5	go to Step 2, 25~35 cycles
	Step 6	72 ℃
	Step 7	Pause at 4℃

Double Digestion (40μL System): (1) Mix the following in a PCR tube:
      Enzyme A    2μL
      Enzyme B    2μL
      10Xbuffer     2 or 4μL (depend on the buffer used)
      BSA              0 or 4μL (depend on the buffer used)
      DNA             20μL (insert DNA) or 10μl (vector DNA)
      dd water      up to 40μL
(2) Incubate at appropriate temperature (30 or 37℃, depend on the enzymes used) for 8~10h (shorter time if the enzymes used have star activity).
(3) Pause at 4 ℃.
(4) Gel running and purify the digestion product.

Agarose gel electrophoresis: (1) Weigh agarose powder according to the volume of the gel wanted (0.1%) and measure the volume of 1×TAE. Add them to a flask.
(2) Melt the mixture in a microwave until the solution becomes clear.
(3) Let the solution cool to about 50℃. Add corresponding volume of DuRed (1:10000, V/V) and mix thoroughly.
(4) Pour the solution into the gel casting tray with appropriate comb.
(5) Let the gel cool until it is solid.
(6) Pull out the comb and place the gel in the electrophoresis chamber carefully.
(7) Add enough 1×TAE to ensure that there is a thin layer of buffer above the surface of the gel.
(8) Pipette DNA samples mixed with appropriate amount of loading buffer and DNA marker into wells on the gel.
(9) Run the gel at 135V for about 20min.

Gel purification with Axygen Gel Extraction Kit: (1) Excise the DNA fragment from the agarose gel with a clean shaver blade.
(2) Weigh the gel slice in a 1.5mL microcentrifuge tube and add 3 volumes of Buffer DE-A to 1 volume of gel.
(3) Incubate at 75°C for 10min (or until the gel slice has completely dissolved). To help dissolve gel, mix by inverting the tube several times every 2~3 min during the incubation.
(4) Add a half volume of Buffer DE-B to 1volume of Buffer DE-A.
(5) Add a volume of isopropanol to 1 volume of the gel if the DNA fragment is shorter than 400bp.
(6) Apply the solution from the last step to the column.
(7) Centrifuge at 12000rpm for 1min and discard the flow-through.
(8) Wash the column by adding 500μL Buffer W1 and centrifuge at 12000rpm for 30sec. Discard the flow-through.
(9) Wash the column by adding 700μL Buffer W2 and centrifuge at 12000 rpm for 30sec. Discard the flow-through.
(10) Wash the column by adding 700μL Buffer W2 and centrifuge at 12000 rpm for 1min. Discard the flow-through.
(11) Centrifuge at 12000rpm for 1min to remove the residual wash buffer.
(12) Place the column in a new clean 1.5mL microcentrifuge tube and add 30μL ddH2O to the center of the column. Stand for 1min (or longer for better elution).
(13) Centrifuge at 12000rpm for 1min (or longer for better elution).
(14) Apply the flow-through from the last step to the center of the column. Stand for 1min (or longer for better elution).
(15) Centrifuge at 12000rpm for 1min (or longer for better elution) and discard the column.

Ligation: (1) Test the concentration of the DNA samples.
(2) Pipet the following into a PCR tube:
      Linearized vector DNA
      Insert DNA
      Solution I
(3) Mix thoroughly and incubate the mixture at 16℃ for 2~3h.
(4) Pause at 4℃.

Tranformatiom: (1) Get the competent cell from -80℃ and wait for its fusion on ice.
(2) Add the entire ligation product or 1μL plasmid into the tube.
(3) Mix and incubate on ice for 30min.
(4) Heat pulse at 42℃ for 90sec.
(5) Put back the tube on ice and incubate for 5min.
(6) Add 1mL LB and incubate at 37℃ with vigorous shaking for 1h.
(7) Centrifuge at 4000rpm for 5min and remove most of the supernatant with about 100μL left.
(8) Resuspend the compact cells and plate the culture on LB plate containing corresponding antibiotics.

Mini-prep with Axygen Mini-prep Kit: (1) Pick a single colony from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic. Incubate for proper time (depending on strains and antibiotic) at 37℃ with vigorous shaking.
(2) Harvest the bacteria cells by centrifuge at 12000 rpm in 1.5mL microcentrifuge for 1min at room temperature and remove all traces of supernatant.
(3) Repeat the last step.
(4) Resuspend compact cells in 250μL buffer S1.
(5) Add 250μL buffer S2 and mix thoroughly by inverting the tubes 4~6 times.
(6) Add 350μL buffer S3 and mix thoroughly by inverting the tubes 6~8 times.
(7) Centrifuge at 12000rpm for 10min.
(8) Apply the supernatant from the last step to the column.
(9) Centrifuge at 12000rpm for 1min and discard the flow-through.
(10) Wash the column by adding 500μL buffer W1 and centrifuge at 12000rpm for 1min. Discard the flow-through.
(11) Wash the column by adding 700μL buffer W2 and centrifuge at 12000rpm for 1min. Discard the flow-through.
(12) Repeat the last step.
(13) Centrifuge at 12000rpm for 1min to remove the residual wash buffer.
(14) Place the column in a new clean 1.5mL microcentrifuge tube and add 30μL ddH2O to the center of the column. Stand for 1min (or longer for better elution).
(15) Centrifuge at 12000rpm for 1min (or longer for better elution).
(16) Apply the flow-through from the last step to the center of the column. Stand for 1min (or longer for better elution).
(17) Centrifuge at 12000rpm for 1min (or longer for better elution) and discard the column.

Preparation of competent cells

(1) Pick a single colony from a selective plate and inoculate a culture of 20mL LB medium. Incubate overnight at 37℃ with vigorous shaking.
(2) Add 2mL of the incubated bacteria solution into 50mL LB medium. Incubate for about 1.5h by shaking at 220rpm until OD600 grows to between 0.5 and 0.6.
(3) Incubate on ice for 15min. Harvest the bacteria cells by centrifuge at 6000rpm for 5min at 4℃.
(4) Remove all traces of supernatant and add 10 ml precooled buffer I on ice. Shake slightly to resuspend the cells.
(5) Put the tube on ice for 2h. Harvest the bacteria cells by centrifuge at 6000rpm for 5min at 4℃.
(6) Remove all traces of supernatant and resuspend the cells with 3.5mL precooled buffer II.
(7) For every 100μL, separate the solution into precooled sterile Eppendorf tube.
(8) Store at -80℃ after liquid nitrogen freezing.

Buffer I:	100mM RbCl	1.2g/100mL
	45mM MnCl2·4H2O	0.891g/100mL
	30mM KAc(pH 7.5)	0.294g/100mL
	10mM CaCl2·2H2O	0.147g/100mL
	glycerol (pH 6.8)	15% (w/v)

Buffer II:	10mM MOPS	0.209g/100mL
	10mM RbCl	0.12g/100mL
	75mM CaCl2·2H2O	1.1025g/100mL
	glycerol (pH 6.8)	15% (w/v)

Measuring GFP expression with ELISA: (1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.
(2) Incubate at 37℃ for 20h with vigorous shaking.
(3) Sample 1mL of the bacteria solution every 2h until 10h and sample 1mL of the bacteria solution after 20h since the start of the incubation.
(4) Measure the fluorescence (excitation: 485nm, detection: 535nm)

Observation GFP expression with Confocal Microscope: (1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.
(2) Incubate at 37℃ for 20h with vigorous shaking.
(3) Sample 1mL of the bacteria solution every 2h until 10h and sample 1mL of the bacteria solution after 20h since the start of the incubation.
(4) Observation GFP expression with Confocal Microscope.

Drawing growth curve: (1) Prepare the mother solution of atrazine (200mmol/L) with ethanol.
(2) Dilute the mother solution with LB into four different concentrations of atrazine: 200μmol/L; 500μmol/L; 1000μmol/L.
(3) Take 52 15mL tubes, divide them into four groups: Group 0, Group 200, Group 500 and Group 1000, 14 tubes each.
(4) Label number 0-13 on each tube in each group. (Each group has tubes labeled 1-13.)
(5) Add 10mL diluted atrazine solutions into Group 0, 200, 500 and 1000. 0μmol/L atrazine solution for Group 0, 200μmol/L atrazine solution for Group 200, 500μmol/L atrazine solution for Group 500 and 1000μmol/L atrazine solution for Group 1000.
(6) Add 1μL Bacteria culture (K12) into each tube, and put them all together into the shaker.
(7) Set the speed of the shaker to 200rpm.
(8) Take corresponding tubes out of the shaker every 1h. For example, tubes which are labeled 4 should be taken out after four hours, tubes which are labeled 13 should be taken out after 13 hours.
(9) Test the OD of the solution in the tubes immediately. Each tube has to be tested for three times. (Use the bacteria-free atrazine solution with corresponding concentration as control.)
(10) Record the data and analyze them.

HPLC: (1) Prepare the mother solution of atrazine (100mmol/L) with ethanol.
(2) Dilute the mother solution with LB into 500μmol/L.
(3) Take 24 15mL tubes, divide them into 8 groups: 0, K12, GFP, TRM, TrzN-, TrzN+, TRM+TrzN-, TRM+TrzN+, 3 tubes each.
(4) Add 10mL 500μmol/L atrazine solution into each tube.
(5) Do nothing to Group 0; add 1μL bacteria culture with corresponding features into each group of tubes.
(6) Put all the tubes into the shaker, set the speed to 200rpm.
(7) Add 12μL solution IPTG into Group TrzN+ and Group TRM+TrzN+ to induce the expression of TrzN after 5 hours of culture.
(8) After 24h of culture, take all the tubes out and put them on the ice.
(9) Treat all the tubes with centrifuge for 10000rpm, 2min.
(10) Filtrate the 1mL supernate in each tube with 0.22μm-filter membrane into EP tubes.
(11) Analyze the samples with HPLC.

Verification of the motility from phenotype: (1) Pick a single colony with required plasmids from a selective plate and inoculate a culture of 5mL LB medium containing the appropriate antibiotic.
(2) Incubate at 37℃ for appropriate time with vigorous shaking until the bacteria reach their plateau phase.
(3) Add 5μL bacteria solution from the last step into 5mL LB medium and incubate at 37℃ for 3h with vigorous shaking.
(4) Inoculate 2μL suspension from the last step on semisolid (the concentration of agar or agarose depends) LB plate.
(5) Incubate at 37℃ for 20h.
(6) Take photos with Gel Analysis System.