Add 1g MgCl₂·6H₂O into 15 mL aseptic centrifuge test tube and increase the volume to 10 mL, then get the MgCl₂·6H₂O solution.

0.5 g PEG8000, 16.0 mL H₂O, 1 mL MgCl₂·6H₂O solution and 2.5 mL Tris-HCl (1 M, pH=7.5) are added into 50 mL aseptic centrifuge tube. It can be mixed with vigorous shaking (can also be dissolved by heating at 60°C), divided into 4 mL in 5 mL aseptic centrifuge tube to obtain Tai Chi buffer, stored at -20°C.

4 mL of pre-cooled Tai Chi buffer are added with 1.4 μL (or 0.8 μL, slightly lower efficiency) NEB T5 exonuclease, mixed and divided into 16 μL in PCR tube to obtain Tai Chi Mix (1.25 x) and stored at -20°C.

It is recommended to use Takara PrimeSTAR® Max DNA Polymerase (R045B), specific primer Tm260C, and 20 μL PCR system to add 0.1~0.5 ng plasmid template to reduce false positives and increase amplification speed (plasmid template pre-diluted to 0.5 ng/μL), extended for 1 min to amplify 7k fragments. With overlapping sequences at least 8 bp, the efficiency of 12 bp is significantly improved, and the efficiency of 20 bp is unchanged. High GC (80%) is extremely inefficient, low GC (10%) is slightly less efficient, and medium GC (50%) is the most efficient.

Take 2 to 12 unpurified DNA fragments that need to be assembled, Mix them with 5 μL each (arbitrary volume, arbitrary proportion), take 4 μL mixed fragments, add them to 16 μL Tai Chi Mix, mix them at 40°C, 60 min (5 fragments, 10 min). Ice bath for 5 min, take 5- 10 μL to transform E. coli (Top10 and DH5α with the highest efficiency), and coating glass beads (Tiangen, GB101-01).

(1)Generating a Linearized E. coli Cloning Vector

• Prepare the linearized E. coli cloning vector using restriction enzymes (single or double digest) or using PCR amplification. When generating the linearized vector by restriction digest, we recommend that you digest the vector with two restriction enzymes rather than a single enzyme to reduce the amount of background.

• Use of restriction enzymes that leave 3’ protruding, 5’ protruding, or blunt ends to linearize your cloning vector are all compatible with this kit. However, for best results we recommend use of a restriction enzyme that produces blunt or 3’ protruding ends when possible for achieving maximum cloning efficiency.
It is very important to have a complete digest (i.e., very low background of uncut vector). Therefore, we recommend increasing the enzyme digestion time (2–3 hours to overnight) and the reaction volume. Double digestion with two restriction enzymes is the most efficient way of linearizing your cloning vector. A double digest followed by PCR amplification of your linear vector virtually eliminates any background.

• Analyze your restriction digestion products using agarose gel electrophoresis to verify that the digest is complete and then purify the digested vector.

(2)Preparing DNA Inserts by PCR

• Seamless Cloning reaction requires that each DNA fragment share a 15-25 bp (base pair) end-terminal homology with the adjacent fragment (including the cloning vector). Therefore, PCR primers used for generating inserts must have 15- nucleotide overhangs on their 5’ ends to provide this homology with the adjacent fragments; however, this homology may be split between the primers used for adjacent PCR-amplified DNA fragments.

• Restriction enzyme-treated vectors can have 5´-overhangs, 3´-overhangs or blunt ends. When vector is linearized by restriction digestion, the entire overlap sequence must originate from the vector sequence and must be added to primers that will be used to amplify the insert. The overlap region of the forward primer for the gene of interest should line up with the 3´end of the overhang on the vector’s left arm and extend back until the Tm ≥48°C. This primer also includes gene-specific sequence at the 3´-end. PCR primers should be up to 40 nucleotides in length (15 nucleotides to provide the requisite homology at the 5’ end and 18–25 nucleotides specific to your DNA element).
It should be noted that the restriction site, which was used to digest the vector, will be lost in the assembled product. However, additional nucleotides may be added between the overlap region and gene-specific sequence region to restore the pre-existing restriction site, or to introduce a new, unique restriction site. A similar principle is applied to the design of the reverse primer for the gene of interest.

(3) Performing the Seamless Cloning Reaction

• Set up the following reaction on ice:

• Incubate at 50°C for 20 minutes. After the reaction, the tube is placed on ice for 2 min immediately.

•Transform 4 µL of the reaction mixture into competent E. coli, or use the mixture directly in other applications. A double digest followed by PCR Component Volume.

Preparations: Check whether RNase A has been added to Buffer P1; Check whether absolute ethanol is added to the Wash Solution. Buffer P2 and P3 were checked to avoid precipitation.

Take 2 mL bacteria cultured overnight, centrifuge at 8000 x g for 2 minutes, and pour the medium. If there is still a large amount of medium left, use a pipetting gun to suck out the remaining medium in the centrifuge tube and discard it.

Add 250 μL Buffer P1 to the precipitate to completely suspend the bacteria.

Add 250 μL Buffer P2, and gently reverse the centrifuge tube 5-10 times then mix. Let stand for 2-4 minutes at room temperature.

Add 350 μL Buffer P2, and gently reverse the centrifuge tube 5-10 times then mix.

Centrifuge at 12000 x g for 5-10 minutes.Transfer the supernatant to the adsorption column, centrifuge at 8000 x g for 30s, and decant the liquid in the collection tube.

Add 500μL Buffer DW1, centrifuge at 9000 x g for 30s, and pour out the liquid in the collection tube.

Add 500μL Wash Solution, centrifuge at 9000 x g for 30s, and pour out the liquid in the collection tube. The above operations should be done twice in a row.

The empty adsorption column was centrifuged at 9000 x g for 1 min and dried at 60 °C for 5 min.

Put the adsorption column into a clean 1.5 mL centrifuge tube, add 40 μL ddH₂O to the center of the adsorption film, leave it at room temperature for 1 minute, and then centrifuge at 9000 x g for 1 minute. Finally, the DNA solution in the tube has been saved.

Select the appropriate horizontal electrophoresis tank and adjust the plane of the electrophoresis tank to the horizontal. Check the line between the regulated power supply and the positive and negative electrodes.

A dot comb with an appropriate pore size is selected and stood vertically on one end of the electrophoresis gel mold, so that the distance between the bottom of the dot comb and the bottom of the electrophoresis gel mold is 1.0 mm.

The appropriate concentration of agarose gel is selected and prepared according to the molecular weight of the sample to be electrophoresed. The gel is heated at 100 ℃ until the agarose melted uniformly.

amount of agarose gel solution is taken with a straw to seal the sides of the electrophoresis gel mold to A small prevent penetration when watering the agarose gel plate. Pour gel to avoid bubbles and try to suck carefully with pipettes if they still remain.

After the agarose gel had set, it is left at room temperature for 20 min, and the spot comb and baffle at both ends of the electrophoresis gel mold are carefully pulled out to keep the spot hole intact.

The electrophoresis gel mold is placed into the electrophoresis tank, and the electrophoresis buffer is added so that the electrophoresis buffer level is 1-2 mm above the surface of the agarose gel. If there are bubbles in the sampling hole, carefully suck out with a straw to avoid affecting the sample.

DNA samples are mixed with 1/5 volume of bromophenol blue indicator spotting buffer. Loading buffer can not only improve the density of the sample and make the sample sink evenly into the sample well, but also make the sample color, which is convenient for loading and estimating the electrophoresis time and judging the position of the electrophoresis.

The samples are carefully added into the sampling wells with a micropipettor, and the sample spot sampling sequence is recorded.

Cover the electrophoresis tank, turn on the power switch, the maximum voltage does not exceed 5 V/cm(100-150 V constant pressure electrophoresis), so that the DNA moves from negative to positive electrode.

The electrophoresis time varies with the specific requirements of the experiment. After the electrophoresis is completed, the power is turned off, the gel is removed with disposable gloves, all the electrophoresis buffer is dried as far as possible, and the results are observed and recorded under a projection UV lamp.

Prepare and perfuse the separation gel and concentrated gel.

The protein marker and sample are successively added to the gel wells with a pipetting gun, and the sample amount (mainly the quality) is consistent in each well.

After the gel and glass plates are placed in the electrophoresis buffer, the voltage and time can be set to begin the electrophoresis. Electrophoresis is stopped when the tracer dye crossed the gel.

After electrophoresis, the gel is transferred to a clean glass or plastic container. Five times 0.25% Coomassie Brilliant blue R250 of the gel is added and incubated at room temperature on a shaker.

Prepare the necessary developing solvent. The volume ratio of trichloromethane to methanol in the mixture is 10:1.

Divid the 5 mL developing solvent to both unfolding cylinders on average.

Draw a horizontal line approximately 0.5 cm from the bottom of the prepared thin layer plate. -And one site is marked every 0.5 cm on the line, for a total of four sites on one plate.

Use capillary tubes to draw the sample and drop it onto the site previously drawn, taking care to make several times to avoid mixing two adjacent samples together.

Place the plate after sampling in the unfolding cylinder and remove it when the unfolding agent is about to reach the top.

Use the tweezers to clamp the unfolded plate and make it touch the cerium molybdate chromogenic agent adequately.

The dyed samples were dried with the heat gun, and the samples could be observed qualitatively after drying.

Culture the E. coli with recombinant plasmids at 37°C to an OD₆₀₀ value of 0.6 to 0.8

Add 0.025 mL the inducer isopropyl-β-D-thiogalactoside (1 mmol/L IPTG, final concentration is 0.5 mmol/L)

Add 0.32 mL 5-aminolevulinic acid (100 mmol/L ALA, final concentration is 0.64 mmol/L )

Add 0.15 mL of ferric chloride (100 mmol/L FeCl₃, final concentration is 0.3 mmol/L)

Continue to culture at 25°C for 24-32 h

Collect the fermentation broth at the end of the previous induction step

Measure the biomass OD₆₀₀ and control the OD₆₀₀ (obtain 2mL finally with OD₆₀₀=30)

Centrifuge a certain amount of the fermentation broth at 8000 rpm for 5 min at 4°C

Collect the E. coli cell and wash with potassium phosphate buffer at pH 8.0

Resuspend the washed cells in potassium phosphate at pH 8.0 containing 5% to 10% v/v of glycerol

Add the integrants respectively (as shown in the table) to the 24-well plate, make them react at 220 rpm and 30°C for 12 h

Add 2 mL ethyl acetate, shake for about 5 s and entrifuge at 8000-9000 rpm for 3 min to obtain the first extraction solution

Take the supernatant into a 2 mL centrifuge tube and dry

Then add another 2 mL ethyl acetate to the precipitate as described above

Take the above samples, add 200 μL extractant (acetonitrile: methanol =4:1) and shake for 1 min, followed by centrifugation at 14000 rpm for 20 min

Detect the product 6β-ΟΗ DCA by high-throughput methods

Collect 1 mL of the fermentation broth at the end of the previous induction step and centrifuge at 14000 rpm for 10 min at 4 °C.

Take 0.2 mL of the supernatant and mix with 0.2 mL of ammonia and 1.6 mL of acetonitrile.

Collect the supernatant by centrifugation at 14000 rpm for 20 min to detect extracellular heme.

Resuspend the precipitated cell pellet in 0.2 mL ultrapure water, 0.2 mL ammonia water, and 1.6 mL acetonitrile.

Place the samples on ice and the cell suspension was sonicated with an ultrasonic crusher for 10 min (power 30 W, 3 mm conical microtip probe, pulse cycle 3 s on / 3 s off).

The supernatant was collected by centrifugation at 14000 rpm for 20 min and used to detect intracellular heme.

Utilize liquid chromatography for heme detection.

The abscissa (different concentrations of substrate) and ordinate (different peak areas) are used to establish standard curves and obtain specific formulas to establish the relationship between concentration and peak areas.