• For "intramolecular" ligation of a PCR product the 5'-ends are phosphorylated with T4 Polynucleotidekinase (PNK)
• Set up the following reaction:

reagent	volume (in µL)
10x T4 DNA ligase buffer	2.5
T4 PNK	1
PEG 4000 50%	2.5
DNA (100 to 600 ng)	x
water	to 25

• Incubate at 37 °C, 30 min
• Heat inactivate at 65 °C, 20 min
• Add 1 µL T4 DNA ligase after reaction has cooled down to room temperature
• Incubate at room temperature for at least 2 h, overnight also works.
• Next step: Transformation via heat shock

Preparation of amino acids

• According to protocol from Caschera and Noireaux 2015b. Weight 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 weight (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

Preparation of cofactor premix

• Stock solutions needed:
• 2 M HEPES
• 174 mM NAD
• 33.9 mM folinic acid
• 65 mM coenzyme A (CoA)
• 50 mg/mL E. coli tRNA (Roche)
• 200 mM putrescine
• 1.5 M spermidine
• Combine stock solutions to a create cofactor premix that is 20x final reaction concentration, depicted in the following list
• 1 M HEPES
• 6.6 mM NAD
• 1.4 mM folinic acid
• 5.4 mM coenzyme A (CoA)
• 4 mg/mL E. coli tRNA (Roche)
• 20 mM putrescine
• 30 mM spermidine

Cell harvest

• 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 protocol section).
• harvest protocol - keep everthing on ice between the steps!
1. Transfer culture into prechilled and weighted harvest tubes or falcons
2. Centrifugate: 5000x g, 4 °C, 15 min. While centrifugating, add DTT to S30 buffer to a final concentration of 2 mM
3. Discard supernatant and weight pellets
4. 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.
5. Centrifugate: 5000x g, 4 °C, 12 min
6. Discard supernatant
7. Repeat steps 4 to 6 two times
8. Centrifuge a last time at 5000x g, 4 °C, for 5 min and remove residual washing buffer by pipetting.
9. Flash freeze pellets in liquid nitrogen and store at -80 °C, unless you want to proceed with sonification directly.

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• 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

• Thaw 100 µl chemo competent E. coli cells on ice
• Add 0.5-5 µl plasmid to 100 µl chemocompetent cells
• Store cells on ice for 10-30 min on ice
• Heat shock for 90 seconds at 42 °C
• Store reaction on ice for 60 seconds
• Optional: Preheat SOC medium to 37 °C
• Transfer reaction to 1 ml SOC medium and incubate at 37 °C for at least 1 hour
• Centrifuge 3 minutes at 12000 rpm and plate on selective LB medium
• Incubate at 37 °C over night

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