Team:WHU-China/Notes

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Materials for cloning all the genes

Bacteria strain:

E.coli DH5α

Minimal Medium:

M9:

For 1L Medium add

Na₂HPO₄·12H₂O 15g

KH_{2PO4 3g}

NaCl 1g

NH_{4Cl 0.5g}

After autoclaving, add:

MgSO_{4(1mol/L) 2mL}

Bacteria strain:CaCl_{2 100μL}

Triton X-100 (as emulsifier) 2uL

Note：

1.For M9 medium using oleic acid as sole carbon source, various amount of oleic acid was first emulsified 1:1 with 10% Triton X-100. M9 medium was then slowly added with constant vortex. M9 medium with high concentration of oleic acid was diluted by M9 medium with triton to form various concentrations；

2. For M9 medium using glucose as sole carbon source, M9 medium with high concentration of glucose was diluted by M9 medium to form various concentrations.

Step:

1. Seed liquor which was activated over night was inoculated into M9 medium which contains different concentration of oleic acid. And it was then incubated at 37℃ for 24 hours;

2. After 24h of incubation in 24 well plates in 37℃, bacteria culture was centrifuged at 3000rmp for 5min, washed and resuspended in PBS;

3. We detected the OD600 and fluorescence of using SpectraMax M2 plate reader (Molecular Devices) .Excitation at 584 nm and emission at 607 nm were used. All fluorescence was normalized with cell density by measuring the absorbance at 600 nm.

Result:

Normalized using Fluorescence/0D600

Blue: Constitutive promoter J23110

Red: PfadR

Glucose Concentration gradient: 0.5, 1, 5, 10 mM

Fatty acid Concentration gradient: 0.025, 0.05, 0.1, 0.25, 0.5, 1, 1.5 mM

Protocols of Cupric-Soap Reaction:

To test metabolism of long fatty acids, we used oleic acid as sole carbon source in mediums, and used cupric-soap reaction to determinate oleic acid concentration preliminarily.

Modified M9 minimal medium with emulsified oleic acid as sole carbon

Minimal medium was the same with that in Materials and methods for PfadR

What's Important:

Slowly pour M9 minimal medium into mixture of oleic acid and Triton X-100 to get more homogeneous solution.

Analysis the concentration of the oleic acid in the medium by cupric-acetate method

1.Collect 5 ml medium which has been used to cultivate bacteria. Then centrifuge the medium at 3000rpm for 10 min to separate bacteria and medium;

2.Decant 3ml supernatant liquid into a 10ml EP. Add 3ml acetone to the liquid, 1ml at a time, shaking 10-20 times before adding another 1 ml in order to avoid the effect of the ions of the liquid during the extraction progress;

3.Add 3 ml isooctane once ,shaking for at least 90s, stand for 2min or longer until layering completely;

4.Collect 3 ml clear isooctane in a 5 ml EP, Add 800l cupric-acetate (5% m/v, adjust pH to 6.8 with pyridine), Shaking for 90 seconds, stand for 2min or longer until layering completely;

5.Detect OD of organic phase in a spectrometry at 715nm.

The Standard Curve:

We add 1.6ml, 3.2ml, 4.8ml, 6.4ml, 9.6ml, 11.2ml oleic acid into 3ml M9 medium to generate a gradient. The absorbency is measured as described in the protocol.

We slightly modified the methods provided by Kwon and Rhee, 1986, adding the extraction step, for it is dispensable when bacteria are in the medium. However, the standard curve is still very close to the paper’s, which proves that plausible and accurate

Krebs-Ringer phosphate（KRPD buffer solution）：

Solution A：

For 100 mL:

NaCl 7.5985g ; KCl 0.3727g ; CaCl2 0.1054g; glucose-H2O 0.9495g,

For 100 mL:

NaH2PO4·2H2O 0 .2964g ;Na2HPO4·12H2O 2.9011g，

After dissolving, add MgSO4·7H2O 0.3130g

Mix A solution and B solution, add 790ml dd H2O, then regulate pH to 7.2~7.4 using 1M NaOH/1M HCl, finally add dd H2O to a final total volume of 1L.

Procedures

1. cultivate cells in 1 liter of medium to late exponential phase;

2. harvest by centrifugation at 35℃;

3. wash twice with 2 liters of warm(35℃)0.05M potassium phosphate buffer (pH 7.4) containing 1%(v/v) Triton X-100;

4. resuspend in 0.05M potassium phosphate buffer (pH 7.4) containing mercaptoethanol;

5. add sufficient volume of buffer to give a concentration of about 50mg(dry weight) of cells/ml;

6. disrupt cells with a Bransor Sonifier for 1min. The treatment was applied for 15s intervals, under 4 ℃;

7. centrifuge at10,000×g for 30min;

8. decant supernatant liquid for enzymatic assay, the protein concentration was determined by the biuret method;

9.analyze fatty acid oxidation in cell-free extracts

Note: little oxidation was observed when less than 1mg.

Reaction mixture:

1.0ml of freshly oxygenated Krebs-Ringer phosphate(pH 7.4)

Palmitate-1-14C, 20nmole (80,000 counts/min)

CoA, 1 μ mole

NAD, 1μ mole

ATP, μ mole

Succinate, 10 n moles

Supernatant protein, 1~5 mg

dd H2O to a final total volume of 2.0ml

Cellulose biosynthesis

1. Inoculate the bacteria into liquid LB medium and then incubate it for 24 hours at 37℃;

2. After centrifugation, supernatant is preserved for use, while deposits are resuspended with PBS and adjust to OD600 1.0;

3. Exceed cellulase was used to digest cellulose in supernatant and deposits. After the cellulase is added, 1mL solution was sampled every 3 min and cellulose of the sample was inactivated immediately;

4. Ten minutes later, the reduce sugar is detected in all the samples with or without cellulose;

5. 2mL DNS solution is mixed with the sample, and incubated in boiling water for two minutes, then it is cooled rapidly;

6. After 9 mL ddH2O being added into the solution, record absorbance at 540nm;

The Standard Curve of glucose concentration

Materials and methods of SDS-PAGE:

Coomassie Blue Stain

-Coomassie brilliant blue G-250 50mg

-95% ethanol 25ml

-85%H3PO4 50ml

-H2O, adjust to 500ml

-filter

Destain solution

-methanol 250ml

-acetic acid 50ml

-H2O adjust to 500ml

2x SDS loading buffer

-0.5mol/l Tirs-HCl(PH6.8) 25ml

-10%SDS 8ml

-50%glycerol 20ml

-2-mercaptoethanol 2ml

-1%Bromphenol Blue 4ml

-H2O adjust to 100ml

10xSDS-PAGE running buffer

Tris base, 30.3 g

M glycine 144.1g

SDS 10 g

-H2O adjust to 1L

Steps

Protein expression

1.inoculate the liquid strains into LB medium supplement with 50μg/mL ampicillin, incubate the medium at at 37℃ until OD600 reaches 0.6;

2. Separate the culture into two test tubes. Add IPTG into one of two tubes at a final concentration of 1mM to induce the expression of

3. 1 mL of the culture is sampled every hour. After centrifugation, deposits was suspended with 300 μL PBS and 200 μL 2x SDS loading buffer, then incubated in boiling water for 15 min.

4. 10μl of samples was load in lanes, run the SDS-PAGE

5. Stained the SDS-PAGE 5 hours

6. Destain the SDS-PAGE until you can see the protein band.

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