Revision as of 08:48, 22 August 2015

Thaw 50 µl electrocompetent E. coli cells on ice, dilute with icecold 50 µl glycerine (10%) if necessary
Add 0.5-5 µl plasmid to 50 µl electrocompetent cells
Store cells on ice for 1 minute
Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ω
Transfer transformation reaction to 450 µl SOC-Medium and incubate 1 h at 37 °C
Plate on selective LB-Medium
Incubate over night at 37 °C

2015 InterLab protocols

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According to protocol from Caschera and Noireaux 2015b. Weigh all aminoacids seperatly into microcentrifuge tubes. Add 500 µl of 5 M KOH to each amino acid. Solubilization is achieved via multiple inverting and, if necessary, vortexing. Especially tyrosine takes a while, and is a suspension rather than a solution. Stock solutions are afterwards stored at -20 °C. Note: According to Caschera and Noireaux, these stock solutions can only be stored a few weeks.

	Molecular weight	mass to weigh (in mg) to obtain desired stock-solution	concentration in stock solution in mM
Alanine	89.09	182	4089
Arginine	174.20	202	2314
Asparagine-Monohydrate	150.14	282	3759
Aspartic acid	133.10	250	3752
Cysteine	121.16	149	2465
Glutamic acid	147.13	269	3655
Glutamine	146.15	183	2501
Glycine	75.07	158	4210
Histidine	155.15	255	3281
Isoleucine	131.18	247	3765
Leucine	131.18	167	2549
Lysine	146.19	175	2392
Methionine	149.21	186	2491
Phenylalanine	165.19	142	1716
Proline	115.13	224	3883
Serine	105.90	209	3953
Threonine	119.12	229	3853
Tryptophan	204.23	170	1661
Tyrosine	181.19	217	2396
Valine	117.15	152	2595

Combine stock solutions to an amino acid mixture like depicted below. Add water to 4 mL and 110 µL of glacial acetic acid to adjust pH to about 6.5. Aliquot (do not forget to mix properly!) and flash-freeze in liquid nitrogen, store at -80 °C.

	Volume (in µL) for mixture	Concentration in mixture (without water and glacial acetic acid added)	concentration in final mixture
Alanine	13.6	146	13.56
Arginine	22.2	135	12.53
Asparagine-Monohydrate	13.6	134	12.44
Aspartic acid	13.6	134	12.44
Cysteine	22.2	143	13.28
Glutamic acid	13.6	130	12.07
Glutamine	22.2	145	13.46
Glycine	13.6	150	13.93
Histidine	13.6	117	10.86
Isoleucine	13.6	134	12.44
Leucine	22.2	148	13.74
Lysine	22.2	139	12.91
Methionine	22.2	145	13.46
Phenylalanine	34	153	14.21
Proline	13.6	138	12.81
Serine	13.6	141	13.09
Threonine	13.6	137	12.72
Tryptophan	34	148	13.74
Tyrosine	22.2	139	12.91
Valine	22.2	151	14.02
sum	381.6	2807	260.62

Combine stock solutions to a create cofactor premix that is 20x final reaction concentration, depicted in the following list

The following harvest protocol mainly orientates to the procedures in Sun et al. 2013 and Kwon and Jewett 2015.
Gather all materials needed: Harvest tubes and falcon tubes, washing buffer, DTT aliquot, liquid nitrogen, and a lot of ice. Start the harvest when E. coli culture reaches mid- to late exponential growth phase. For the E. coli we used in our experiments and cultivations, the mid- to late exponential growth phase was reached at an OD₆₀₀ of 3-4 (see growth curves in notebook section).
harvest protocol - keep everything on ice between the steps!

Transfer culture into prechilled and weighted harvest tubes or falcons
Centrifugate: 5000x g, 4 °C, 15 min. While centrifugating, add DTT to S30 buffer to a final concentration of 2 mM
Discard supernatant and weigh pellets
Add about 10 mL of S30 washing buffer and resuspend cells by vortexing and vigourous shaking. For us, cycles of 15 s vortexing/shaking and 30 s resting on ice worked well.
Centrifugate: 5000x g, 4 °C, 12 min
Discard supernatant
Repeat steps 4 to 6 two times
Centrifuge a last time at 5000x g, 4 °C, for 5 min and remove residual washing buffer by pipetting.
Flash freeze pellets in liquid nitrogen and store at -80 °C, unless you want to proceed with sonification directly.

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After every step the reaction tube is centrifuged with 1000 g for 1 minute.
The supernatant after centrifugation and every wash step (starting from the step when the protein is added to the agarose) is stored for analyzing the samples for protein and DNA amounts.
As negative controls no protein to the agarose were added.
Steps:

25 µL Ni-NTA agarose is put in a reaction tube. Then the sample is centrifuged.
The agarose is washed three times with Kpi buffer (Volume: 50 µL).
250 pmol protein in 20 µL Kpi buffer is added and incubated for 30 min. Then the sample is centrifuged.
The agarose is incubated three times in Kpi Buffer (Volume: 50 µL).Then the sample is centrifuged.
0.75 pmol plasmid (lacO) is mixed with 20 µL binding buffer (Incubation time: 15 min).
Unbound DNA is washed away three times with binding buffer (Volume: 50 µL, Incubation time: 15 min). Then the sample is centrifuged.
3x elution with analytes in binding buffer (Volume: 50 µL, Incubation time: 15 min)
Imidazol solution (50 µl) is used to release proteins from the agarose.
Water (volume: 10 µL) is mixed with agarose.
The DNA amount of the supernatant after centrifugation is analyzed via gel electrophoresis.

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You can detect immobilized DNA und your fusion protein by measuring the fluorescence.
You need Cy3- and amino-labeled DNA with an operator site for immobilization, a repressor fused to a fluorescence protein and cellulose on a black 96-well plate.

Excitation for Cy3: 545 nm, Emission for Cy3: 590 nm
For example: Excitation wavelength for sfGFP: 480 nm, Emission wavelength for sfGFP: 515 nm
The gain needs to be adapted.

After every step the supernatant is transferred into another 96-well plate for measurement.
The plate with the cellulose is dried after having taken out the supernatant and measured again.

Cellulose on the plate is prepared in a black 96-well plate.
DNA is immobilized on cellulose.
Protein in Kpi buffer (Concentration: 20 µg/mL, Volume: 25 µL) is added to the wells with immobilized DNA (Incubation time: 15 min).
The wells are washed with binding buffer (Volume: 200 µL, incubation time: 10 min).
100 µl analyte solution (e.g. 0.5 mM IPTG in binding buffer) is put in the well and shaken for 30 min.

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