LAB NOTE

Motility Control -CheZ-

Assay

09/01
Over night culture of
p01CheZ
A1
were diluted in LB medium 50 times.
Prepared semi solid agar plate with appropriate concentration of ampicillin.
Put two samples on semi solid agar plate (2 plate for each sample).
Set them under time lapse camera.

09/08
Dilluted 1uL of p02CheZ into 1mL semi solid agar.
Took 50ul of agar to cubet and measure OD600 every 10 seconds.

09/09
Dilluted 1uL of JM109into 1mL semi solid agar.
Took 50ul of agar to cubet and measure OD600 every 10 seconds.

09/10
Dilluted 1uL of JM109 into 1mL semi solid agar.
Took 50ul of agar to cubet and measure OD600 every 10 seconds.

09/14
Dilluted 1uL of p02CheZ into 1mL semi solid agar.
Took 50ul of agar to cubet and measure OD600 every 10 seconds.

09/15
Over night culture of
p02CheZ
p01CheZ
p10CheZ
were diluted in LB medium 40 times.
Prepared semi solid agar plate with appropriate concentration of ampicilin.
Put above 4 samples on semi solid agar plate (6 plate for each sample).

09/15
Dilluted 1uL of p02CheZ into 1mL semi solid agar.
Took 50ul of agar to cubet and measure OD600 every 10 seconds.

09/16
Set 4 plate of p02CheZ and 2 plate of p01CheZ under time lapse camera.
Took photos.

09/17
set 6 plate of p10CheZ under time lapse camera.
Took photos.

Growth Rate Control -Barnase-

Assay

09/08
induce [Plac Barnase GFP] and [Plac Barnase] with 100μM IPTG in 2ml LB culture.

Plac Barnase GFP IPTG+ #1~3
Plac Barnase GFP IPTG- #1~3
Plac GFP IPTG+ #1~3
Plac GFP IPTG- #1~3

We measured OD600 at 0, 90, 245, 420min after from IPTG induction. There are significant difference between [Plac Barnase GFP IPTG+] and [Plac Barnase GFP IPTG- ].

09/14
induce [Plac Barnase GFP] and [Plac Barnase] with 100μM IPTG in 2ml LB culture.

Plac Barnase GFP IPTG+ #1~3
Plac Barnase GFP IPTG- #1~3
Plac GFP IPTG+ #1~3
Plac GFP IPTG- #1~3

We measured OD600 at 0, 50, 90,130, 165, 205, 250, 290, 325 minutes after from induction. However, there are not any significant difference between IPTG induced cell and non-induced cell.

09/17
induce [Plac Barnase GFP] and [Plac Barnase] with 100μM IPTG in 2ml LB culture.

Plac Barnase GFP IPTG+ #1~3
Plac Barnase GFP IPTG- #1~3
Plac GFP IPTG+ #1~3
Plac GFP IPTG- #1~3

We measured OD600 at 0, 45, 80,120 minutes after from induction.
There are significant difference between [Plac Barnase GFP IPTG+] and [Plac Barnase GFP IPTG- ].

PROTOCOL

DNA construction

Transformation
Method

1. Put the competent cell on ice and leave

2. Add 10μL of ligation product to 100μL of competent cell.

3. Place on ice for 30 min.

4. Heatshock at 42 [degree] for 45 sec.

5. Place the tube on ice for 3 min.

6. Add 500μL of LB medium and place tube on 37 [degree] for 30 min.

7. Plate 500 μL of the medium and spread well.

8. Incubate the plate on 37 ℃ over night.

Electrophoresis
Method

1. Add 0.5 g of agarose to 50 mL of 1xTAE buffer in Erlenmeyer flask.

2. Wrap the top of Erlenmeyer flask and make some small hole on wrap.

3. Heat it by microwaves until the solution gets transparent.

4. Pour it into a gel makernd set a comb before gelling.

5. After gelling, remove the comb carefully not to break well.

6. Place the gel into tank for electrophoresis and fill 1xTAE over the level of gel.

7. Add 4 μl of 6xLoading buffer into 20 μl of DNA solution and vortex it.

8. Apply 24 μl of sample and 3 μl of ladder solution in each well.

9. Electrophoresis at 100 V for 20-30 min.

10. The band of bromophenol blue reaches 80% of the gel, stop electrophoresis and salvage the gel.

11. Check the band under blue light.

Colony PCR
Method

1. Mix DNA solution and reagent as follows.
   (Add10xF-universal Primer and 10xR-Univarsal Primer at a final concentration of 5 μM. Then add 2xGoTaqMix and MillQ.)

2. Dispense it to each PCR tube for 10 μl.

3. Pick up single colony on plate and dip to the tube.

4. Set to Thermal cycler. (95℃ 5 min, 95℃ 30 sec, 53℃ 30 sec, 72℃ 30-180 sec (step 2 - 4 repeated 30 times), 72℃ 3 min)

5. Check the length of DNA by Electrophoresis.

Miniprep
Material
WizardR Plus SV Minipreps DNA Purification System (promega)

Method

1. Pour 3 ml of LB broth to test tube.

2. Pick up a single colony and put it into the tube.

3. Incubate it at 37℃ for 12~16 hours with the tube shaking.

4. Transfer 1.5 ml of culture fluid to 1.5 ml tube.

5. Centrifuge at 15000 rpm for 1 min.

6. Throw away the supernatant

7. Add the other 1.0 ml of culture.

8. Centrifuge at 15000 rpm for 1 min.

9. Throw away the supernatant

10. Add 250 μl of Cell-Resuspension Sol

11. Vortex it until precipitation dissolved.

12. Add 250 μl of Cell-Lysis Sol.

13. Invert it not to make bubble.

14. Add 350 μl of Neutralization Sol and invert it not to make bubble.

15. Centrifuge at 15000 rpm 10 min.

16. Set a collection tube on column and apply the supernatant to it.

17. Centrifuge at 15000 rpm for 1 min.

18. Add 750 μl of Column Wash Sol and Centrifuge for 1 min at 15000 rpm.

19. Remove the flow through.

20. Add 250 μl of Column Wash Sol and Centrifuge for 1 min at 15000 rpm.

21. Remove the flow through.

22. Centrifuge for 2 min at 15000 rpm to dry the column.

23. Transfer the column to another 1.5 ml tube.

24. Add 50 μl of nuclease-free water and leave it for 1 min at room temperature.

25. Centrifuge for 1 min at 15000 rpm.

26. Measure concentration of the flow though liquid.

Digestion
Material

The restriction enzymes (EcoRⅠ, XbaⅠ, SpeⅠ and PstⅠ) are purchased or provided as support for iGEM Japan from Promega.
Method

1. Mix DNA solution and reagent as follows.(Add 500ng of DNA for Vector DNA or 700 ng of DNA for Insert DNA. Then add 10x buffer, 0.5 μl of each restriction enzymes, and DW)

2. Incubate for 2 hours at 37℃.

3. Add 2 μl of 10x Loading Buffer and Electrophoresis and Gel Extraction.

Miniprep
Method

1. After electrophoresis, slice the band and put the gel slice into 1.5 ml tube.

2. Add 600 μl of Membrane Bind Sol and stand at 60℃ (Vortex every 2 min)

3. Apply to column-set collection tube and Centrifuge for 1 min at 15000 rpm.

4. Add 750 μl of Membrane Wash Sol to the column.

5. Centrifuge for 1 min and remove the flow through.

6. Add 500 μl of Membrane Wash Sol to the column.

7. Centrifuge for 1 min and remove the flow through.

8. Centrifuge for 2 min to dry the column.

9. Transfer the column to another 1.5 ml tube.

10. Add 20 μl of nuclease-free water and leave it for 1 min at room temperature.

11. Centrifuge for 1 min at 15000 rpm.

12. Measure concentration of the flow though liquid.

Ligation
Method

1. Mix the DNA solution and reagent as follows.
   (Add 3 μl of Insert DNA and 1.5 μl of Vector DNA. Then add 2x Rapid Ligation buffer, and 0.5 μl.)

2. Stand it for 15 min at room temperature.

Assay

Semi solid agar preparation

1. Dilute 0.15 g of Agar to 100 mL of LB medium in 200 mL beaker and wrapped with aluminum foil.

2. Autoclave at 121℃ for 5 min.

3. Stir well by magnetic stirrer and allow them cool between 40℃ and 50℃.

4. Add appropriate antibiotics.

Measurement of growth rate in semi solid agar

1. Using semi solid agar for Auto zero.

2. Dilute 1μl of mid log phase culture into 1 mL semi solid agar in 1.5 mL tube.

3. Pipette up and down well.

4. Take 50μl of agar to 50μl cubet and set it on absorptiometer.

5. Measure OD600 every 10 second for more than 10 hours at RT(25℃).

Measurement of growth rate in liquid medium

1. Dilute over night culture 20-40 times into 2 mL LB medium with appropriate antibiotics.

2. Incubate the culture until reach mid log phase (OD600 is between 0.5 and 1.0).

3. Dilute mid log phase culture 10-20 times into 2 mL LB medium with appropriate antibiotics and induction substance.

4. Incubate the culture and measure OD600.

Motility assay

1. Dilute over night culture 20-40 times into 2 mL LB medium with appropriate antibiotics.

2. Incubate it until reach mid log phase (OD600 is between 0.5 and 1.0).

3. Pour semi solid agar to petri dish and dry for 1.5 hours in a clean bench.

4. Put 2μl of mid log phase culture on the center of a semi solid agar plate.

5. Leave the plate at RT(25℃) for about 12 hours for growth.

6. Take time lapse photograph with the interval 20 min at RT.

Analysis of diffusion rate

1. Import every third image of time lapse photos to ImageJ (corresponding to every 1 hour).

2. Measuring the diameter of Petri dish as standard (9mm).

3. Measuring the diameter of each colony.

4. Plot the diameter against time and see if the diameter is linear to time.

5. Using least-square method to determine colony expanding speed (the dimension is [mm/h]).

6. Using following equation to determine the diffusion rate.
   Using following equation to determine the diffusion rate.

Pattern formation assay

1. Dilute over night culture to 20 to 40 times into 2 mL LB medium with appropriate antibiotics and make them grow until reach mid log phase

2. Take appropriate amount of mid log phase culture into 1.5 mL tube.

3. Centrifuge at 15000 rpm for 1 min and throw away the supernatant.

4. Add semi solid agar (cooled to 40℃) and pipette up and down.

5. Move it to 15 mL falcon tube.

6. Add 3 mL semi solid agar and pippete up and down.

7. Pour into petri dish.

8. Shake well and allow agar to spread.

9. Leave the plate at RT(25℃) for several ten hours.

10. Observe the plate to see if some pattern is formed.