Team:NCKU Tainan/Protocol

Protocol

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Dry Cell Weight

Dry Cell Weight ×

1. Preculture the bacteria overnight in 4ml LB with selective antibiotic.
2. Refresh the precultured bacteria in 20 ml of M9 xylose in flask.
3. Culture the bacteria for 24 hr in normal incubator with 177 rpm and 37℃ condition in 24 hr.
4. Measure the optical density of the sample with 600 nm light wavelength, and then diluted the optical density value to 0.25 ,0.5, 0.75, 1 in 4ml of bacteria respectively.
5. Measure the weight of an empty eppendorf.
6. Transfer 4 ml of bacteria to eppendorf, and then centrifuge them at 16000xg for 3 min.
7. Put the eppendorfs in the 60℃ high-temperature oven with cap opened for 12 hr.
8. Measure the net weight of the bacteria after 12 hr.
9. Analyze the dry cell weight and draw the regression line of it.

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Minimal Salts (M9) Medium Xylose Preparation

Minimal Salts (M9) Medium Xylose Preparation ×

M9 medium contains essential salts and nitrogen. It contains 33.9 g/L Na₂HPO₄·7H₂O, 15 g/L KH₂PO₄, 5 g/L NH₄Cl and 2.5 g/L NaCl; suitable for recombinant E. coli strains

• Minimal salts (M9) medium is suitable for non-selective cultivation of E. coli strains for cloning, production of DNA, plasmid DNA and recombinant proteins.
• Suitable for selective cultivation when appropriate antibiotics are added.

Substances		Volume in ml	Volume(M)
M9 salt solution (10X)
	Na2HPO4	100	33.7 mM
	KH2PO4		22.0 mM
	NH4Cl		9.35 mM
	NaCl		8.55 mM
20% xylose		20	0.4 %
1 M MgSO4		1	1 mM
1 M CaCl2		0.3	0.3 mM

1. M9 salt solution (10X)

Na₂HPO₄·2H₂O 75.2 g/L

KH₂PO₄ 30 g/L

NaCl 5 g/L

NH₄Cl 5 g/L

- Dissolve the salts in 800 ml water

- Add water to a final volume of 1 L

- Autoclave for 15 min at 121°C

2. 20% Xylose

- Add 100 g xylose to 440 ml water

- Add water to final volume 500ml

- Autoclave for 15 min at 121°C

3. 1 M MgSO₄

- Dissolve 24.65 g MgSO₄-7H₂O in 87 ml water

- Add water to final volume 100ml

- Autoclave for 15 min at 121°C

4. 1 M CaCl₂

- Dissolve 14.70 g CaCl₂-2H₂O in 94.5 ml water

- Autoclave for 15 min at 121°C

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PRK Toxicity Test

PRK Toxicity Test ×

1. Preculture the bacteria overnight in 4ml LB with antibiotic.
2. Refresh the precultured bacteria in 4ml of M9 xylose in test tube.
3. Culture in the normal incubator for 12 hr with 177 rpm
4. Measure the optical density of the sample with 600 nm light wavelength at the beginning (0 hr) , and also measure them after 12 hr. All samples should be measured in triplicate.

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DNS Reductive Sugar Measurement

DNS Reductive Sugar Measurement ×

DNS solution preparation:

1. Disolve 2.5g of 3,5-Dinitrosalicylic acid (DNS) to 150ml double distilled water.
2. Heat to solution to 45 degree Celsius and add 4g of NaOH. Stir the solution until it is transparent.
3. Add 75g of potassium sodium tartrate and add water to 250 ml.
4. Keep the solution without light exposure. The solution can be used after 7 days.

Principle:

Under base solution, DNS will turn to brown color while reacting with reductive sugar in high temperature. In the specific temperature range, the color will have linear relationship with the reductive sugar concentration.

Calibration:

1. Prepare glucose or xylose water solution to the following concentration: 0, 0.2, 0.4, 0.8, 1.0, 1.2, 1.6, 2 g/L.
2. Take 200 ul of sample, add 200 ul of DNS solution.
3. Heat the sample at 100 degree Celsius for 10mins.
4. Cool down the sample with ice to room temperature.
5. Measure the optical density of the sample with 540nm light wavelength.
6. Draw the graph of sugar concentration with respect to optical density.

Calibration results:

Measurement:

1. Take 200 ul of sample, add 200 ul of DNS solution.
2. Heat the sample at 100 degree Celsius for 10mins.
3. Cool down the sample with ice to room temperature.
4. Measure the optical density of the sample with 540nm light wavelength.
5. Get the concentration of reductive sugar from the Calibration graph.

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Short Term pH Sensing Systen Measurement

Short Term pH Sensing Systen Measurement ×

1. Preculture the bacteria o/n in LB, and prepared 8 different (pH4, 4.25, 4.5, 4.75, 5, 5.5, 6,7) pH value of M9 buffer.
2. Culture the bacteria in 6 ml LB with selective antibiotic to O.D. 0.6 (about 1.5-2hr)


3. Divide 6 ml of bacteria into 8 eppendorfs, and centrifuged them at 1300 rpm for 1 mins in 37 ℃, then discard the supernatant completely.
4. Add 700 μl per different pH value of M9 buffer into 8 different eppendorfs, respectively . And resuspend the cells completely by pipetting.
5. Add 200 μl of bacteria in 96 well (This step need triple repetition.)
6. Measure the O.D.595 nm at the first and the last time point , and the fluorescence value (485-535nm ) at every 180 sec, within 30 mins.

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Long Term pH Sensing System Measurement

Long Term pH Sensing System Measurement ×

1. Preculture the bacteria o/n in LB, and prepared 8 different (pH4, 5, 6, 7) pH value of M9 buffer.
2. Add 1/100 of the bacteria in 20 ml of different pH value of M9 buffer with selective antibiotic.
3. Culture the bacteria in the incubator in 37℃for 24 hr.
4. Measure the O.D. value (595 nm) and the fluorescence value (485-535nm ) at every 1 hr , within 24 hr.

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Total Solution

Total Solution ×

1. Preculture the bacteria overnight by picking up a colony in 4ml LB with antibiotic.
2. Culture the bacteria in 30 ml LB by adding 1/100 of precultured broth, and selective antibiotic to log phase(about 3 hr).
3. Centrifuge them in 22℃ ,3200xg for 5 minutes , then refresh by the M9 medium with 4%xylose and 0.1%LB.
4. Culture the bacteria for 24 hr in both O₂ and 5%CO₂ incubator, and test the O.D. value at every 6 hours.

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Carbonic Anhydrase Activity Assay

Carbonic Anhydrase Activity Assay ×

Materials:

pH meter, enzyme samples, magnetic stirrer, saturated CO2 solution, 20 mM Tris-HCl buffer (pH8.3), 20mL borosilicate glass vial

Method:

• Saturated CO2 solution preparation

Dissolve gaseous CO2 into deionized water (on ice) until it is saturated. (At least 30 min)

• 20 mM Tris HCl buffer (pH8.3) preparation
1. Dissolve 121.14 g Tris in 800 ml deionized water.
2. Add a pH meter into the solution to observe the pH.
3. Slowly add concentrated hydrochloric acid (HCl) solution to reduce the pH to 8.3. Be careful not to add too much at a time, since the pH will change rapidly.
4. Once the desired pH has been reached, top up the solution to 1 L using deionized water.
• Performing Blank Reaction
5. Add 9 mL ice-cold Tris−HCl (20 mM, pH8.3) buffer into a 20 mL borosilicate glass vial with further incubation at 0 °C with stirring.
6. Add 6 mL of ice-cold CO2-saturated solution immediately into the vial.
7. Record the time course, T₀ (in sec) of pH decrease from 8.3 to 6.3. The pH meter must be preset to 0°C and calibrated.
• Performing Test-Sample Reaction
8. Mix 9 mL ice-cold Tris−HCl (20 mM, pH8.3) buffer and 0.2 mL enzyme, transfer the mixture to a 20 mL borosilicate glass vial with further incubation at 0 °C with stirring.
9. Add 6 mL of ice-cold CO2-saturated solution immediately into the vial.
10. Record the time course, T (in sec) of pH decrease from 8.3 to 6.3. The pH meter must be preset to 0°C and calibrated.
11. Calculate CA activity using a Wilbur–Anderson unit (WAU) per milliliter of sample.

Calculation:

Units/ml of enzyme = (T_{0 Average} - T_Average ) (df) / (T_Average )(E)

T = Time (in seconds) required for pH to change from 8.3 to 6.3 as per Unit Definition

df = dilution factor

E= volume (in milliliters) of enzyme used

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Competent Cell Preparation

Competent Cell Preparation ×

For E. coli DH5α,BL21(DE3) and W3100(DE3) competent cell

1. Streak out wild type E. coli on a plate (LB plate without antibiotics) overnight and pick one colony into 3 ml of media (LB) and grow overnight.
2. Transfer 0.4 ml of starter culture into 40 ml of fresh LB and grow culture at 37 ℃.
3. When the OD600 nm up to 0.35, put the cells on ice immediately.
4. Spin the cells at 4℃ for 10 minutes at 4000 rpm.
5. Suspend the pellet on ice carefully with 16 ml chilly Transformation Buffer 1(TFB1).
6. Leave nicely suspended bugs on ice for 10 minutes.
7. Spin the cells at 4℃ for 10 min. at 4000 rpm.
8. Suspend the pellet on ice with 1.6 ml of Transformation Buffer 2 (TFB2).
9. Leave on immediately on ice for 30 minutes.
10. Aliquot 100 μl into 1.5 ml centrifuge tubes and snap freeze immediately with liquid nitrogen.
11. Store the frozen cells in the -80℃ freezer.

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PCR

PCR ×

1. Gently mix the following reaction by pipetting and centrifuge briefly.

	20 μl system	50 μl system
Template	12～20 ng	30～50 ng
Forward primer	1.0 μl	2.5 μl
Reverse primer	1.0 μl	2.5 μl
dNTP	1.6 μl	4.0 μl
10x Buffer	2.0 μl	5.0 μl

Program of KOD DNA polymerase

Temperature	Time	Repeat
94 ℃	3 min.
94 ℃ (Denaturation)	40 sec	25～30 cycles
57.5 ℃ (Annealing)	30 sec
72 ℃ (Extension)	Depend on sequence size (2 kbp/min. for Taq)
72 ℃	5 min.