1. Digestion

A 25 µL master mix was prepared for both of the inserts. The backbone plasmid was already precut, but to cut backbone plasmids EcoRI-HF and PstI would be used (the same volume).

Enzyme Master Mix for the first insert:

4 µL of the master mix was then mixed with 4 µL of the enzyme.

Enzyme Master Mix for the second insert:

4 µL of the master mix was then mixed with 4 µL of the enzyme.

Both digestion mixtures were digested at 37 °C for 30 minutes. The mixtures were then left at 80 °C for 20 minutes to heat kill the enzymes. These steps were performed in a PCR machine.

2. Ligation

In this step it is important to have equimolar amount of cut plasmid backbone and digest product. In our case the length of everything was very similar, therefore the same volumes could be used, however that can always be done. The total reaction volume is 10 µL

Ligation Mixture:

The mixture was then ligated at 16 °C for 30 minutes. Another heat kill around at 80 °C for 20 minutes was then performed.

For the transformation step see the Phusion PCR, Gibson Assembly and Transformation protocol.

1. Phusion PCR

Total reaction volume 50 µL
Amount of plasmid per reaction ~30 pg

Phusion PCR reaction mixture:

PCR reaction conditions:

Steps 2–4: 25 cycles
Lid temperature at 105 °C

2. DpnI Treatment of the PCR Fragment

To the 50 µL PCR reaction mix from step 1 add:

2.4 µL “FastDigest” DpnI
6 µL 10X “FastDigest” Buffer

1) Incubate reaction mixture at 37 °C for 1 hour
2) Purify the DNA in the mixture with “PureLink Quick PCR Purification Kit” (Invitrogen). Milliq was used instead of TE buffer when eluting the DNA from the spin column in the final step of the DNA purification. The optional washing step was skipped
3) Measure the DNA concentration on Nanodrop in units of ng/µL. 2 µL was used for each measurement
4) Store the DNA at -20 °C until further use

3. Gibson Assembly Reaction

1. Mix PCR fragment from the plasmid and the Gibson fragment containing the insert. The total volume is 20 µL:

Mix i) the PCR fragment made from the plasmid and ii) the Gibson fragment containing the gene of interest. Total volume of the Gibson reaction mix is 20 uL :

2. Incubate the mixture at 50 °C for 1 hour

4. Transformation

1. Take out competent cells (we used Escherichia Coli TOP10 for cloning and E. coli BL21 (DE3*) for protein expression from Invitrogen) from the -80 °C and thaw them on ice from approximately 15 minutes.
2. Add 2 µL of the Gibson mixture/Ligation mixture to the competent cells or ~7.5 ng of gBlocks.
3. Incubate on ice for 30 minutes.
4. Incubate at 42 °C for 30 seconds (water bath).
5. Incubate on ice for 5 minutes.
6. Add 200 µL of LB to the mixture and incubate it at 37 °C with 200 rpm for 1 hour.
7. Plate the mixtures on chloramphenicol plates (200 µL 1:1 dilution and 200 µL 1:100 diluted with LB). Beads were used to spread the mixture on the plates (10–20 beads).
8. Incubate plates overnight at 37 °C.

Electroporation

1. Grow overnight culture in 5 mL LB media.
2. Wash with 1 mL 10% glycerol 5 times in cold room.
3. Resuspend in 250µL 10% glycerol. This is enough for 10 electroporation transformations.
4. Put 1 µL plasmid (~10 ng/µL) in 25 µL cells. Transfer to a cold electroporation cuvette.
5. Electroporate with a 1.8 kV pulse. Immediately add 300 µL SOC media. Incubate in 37°C with 180 rpm shaking for 1h. Plate on appropriate antibiotic agar plate.

1. Colony PCR

Total reaction volume 20 µL.

DreamTaq PCR Reaction Mixture:

A master mix containing everything put the DNA polymerase was made. Enzyme was then added to each PCR tube.

PCR reaction conditions:

Steps 2–4: 32 cycles
Lid temperature at 105 °C

2. Gel electrophoresis

Preparation of agarose for gel electrophoresis:

1. Mix 0.5 % TEB with agarose so that the amount of agarose is 0.8 %
2. Microwave the mixture until it starts boiling. DO NOT PUT THE LID ON!
3. Store at 65 °C until further use

Preparation of gels

1. Take 50 mL of agarose per gel into a separate bottle
2. Add 5 µL of SyberSafe per gel into the agarose
3. Pour the agarose into the cassette, put the comb in and let it polymerize for ~30 minutes
4. Put the gel into the electrode chamber and fill it up with 0.5 % TEB buffer
5. Load 12 µL of PCR product into the wells and add 5 µL of 1 kb Generuler
6. Run the gel at 100 V for 30–60 minutes

Expression

1. Transform plasmid in BL21 (DE3*) E. coli cells to use for expression.
2. Inoculate three colonies from transformation plate into 50 mL LB with 30 µg/ml chloramphenicol. Make a negative control without any colony and grow overnight.
3.Take 20 mL of culture in 2 L LB with 30 µg/ml chloramphenicol to make expression cultures, divided into two 1 L E-flasks. Grow at 37 °C 110 rpm for about 3 hours until 0.6 < OD600 < 0.8. Take out negative control sample that is not induced. Add 0.25-1 mM IPTG to induce protein expression and grow at 37 °C 110 RPM for about 4–6 hours.
4. Divide culture into 4 tubes and centrifuge culture at 5000 rpm for 30 min. Discard supernatant and resuspend each pellet in about 20 mL LB. Pour over to tw0 50 mL Falcon tubes and centrifuge 5 min at 7000 rpm. Discard the supernatant. Flash freeze pellet in liquid nitrogen and store at -80 °C.

IMAC Purification

Buffer A, 1 L
The buffer was made with MilliQ H2O. NaOH was added until pH 7.4 and sterile filtered on 0.2 µm filter.

Buffer B, 0.5 L
The buffer was made with MilliQ H2O. Phosphoric acid was added until pH 7.4 and sterile filtered on 0.2 µm filter.

(Day of purification) 1. Thaw the cells (2 pellets) and add 10 mL buffer A to each pellet. Add 20% of EDTA-free protease inhibitor pill to each pellet. Add 500 µL lysozyme (10 mg/ml) and 2 µL DNAse I (238 units/µL). Put on stirring at 4 °C for 40 min.
2. Use a cell disruptor to lyse the cells and pellet for 1 hour at 3800 rfc and run the supernatant through a 0.2 µm vacuum filter.
3. Add the supernatant to Ni2+-agarose column on an ÄKTA protein purification system. 3 mL fractions were collected.
4. Wash the injection tubes by running buffer A for some minutes. Wash column with buffer B and equilibrate with buffer A. Wash with 50-100 mL buffer A until A280 hit baseline. Start eluting gradient to 100 % buffer B in 60 min. Collect 3 mL fractions.
5. SDS-PAGE analysis
a. Run the fractions containing protein on SDS-PAGE to identify fractions holding the desired protein.
b. Load premade gels (4-20% SDS) with 20 µL sample (10µL protein sample and 10µL of Laemmli buffer) as well as 6µL PageRuler-prestained ladder 10-180 kDa (ThermoFisher) and run the gel in running buffer at 200 V for about 30 min.
c. Stain gels in Coomassie blue, destain and document the result.
6. Pool fractions containing purified protein and perform buffer exchange to PBS+500 mM NaCl using Amicon Ultra Centrifugal filters 10 kDa. This should be done before performing activity measurements.

Activity measurements

1. Before activity measurements of the purified enzyme buffer exchange to PBS+500 mM NaCl should be performed using Amicon Ultra Centrifugal filters 10 kDa. 2. The buffers should be freshly prepared. 3. Use plate reader to performed the measurements. 4. The final volume in each well was 75 µL. 65 µL+ 10 µL of protein (enzyme). 5. Substrates were dissolved in DMSO stock solution. 6. Negative control (substrate without protein) and positive control (product without protein) were used. 7. Buffer without protein, substrate or product was used as a blank. 8. Absorbance spectra was in the 200-700 nm range and measurements were taken at 1-15min intervals until activity had been observed.

CsADH-2946
Protein concentration was varied in each well, see specifics for the experiment here