Experiments

1. PCR systems
   

   PCR systems (to amplify the upstream homologous arm)
   Reagent	Volume（μL）
   2× PCR Mix	25
   Primer F (10 μM) （M14685-90-Gib-up-F）	2.5
   Primer R (10 μM) （M14685-90-Gib-up-R）	2.5
   DNA template（The genome of Streptomyces rapamycinicus NRRL 5491）	2
   ddH2O	18
   Total	50

   PCR systems (to amplify the downstream homologous arm)
   Reagent	Volume（μL）
   2× PCR Mix	25
   Primer F (10 μM) （M14685-90-Gib-down-F）	2.5
   Primer R (10 μM) （M14685-90-Gib-down-R）	2.5
   DNA template（The genome of Streptomyces rapamycinicus NRRL 5491）	2
   ddH2O	18
   Total	50

2. Set-up and run the electrophoresis to test for the positive result.
   

   	PCR instrument program set-up.
   Steps	Temperature（℃）	Time
   1	98	3min
   2	98	30sec
   3	55	30sec
   4	72	2min
   5	72	10min
   6	4	forever

   
   PS: Steps 2-4 cycle 30 times

1. Make the agarose gel
   

   Agarose gel
   Reagent	Volume
   1×TAE buffer	50 mL
   Agarose	0.5 g
   nucleic acid dye Gel Red (After place in a microwave oven on high for 2 min and heat until completely melted)	5 μL

2. Mount the gel plate and insert the comb, then pour the liquid gel onto the gel plate, 3-4 mm high, avoiding air bubbles, and solidify at room temperature.
3. After complete solidification, remove the comb and transfer the solid gel to the electrophoresis bath, adding 1 × TAE buffer to completely submerge the gel.
4. Spotting: add each sample in turn to the spotting wells of the gel, ending with 10 μL of DNA Maker, (add samples to prevent touching the gel around the sample wells and penetrating the bottom of the gel).
5. Electrophoresis: cover the electrophoresis tank with the sample addition wells at the negative end of the electric field and turn on the power, 180V, for 15 min.
6. Blue light observation: turn off the power, remove the gel, and observe under the blue light gel illuminator, the red fluorescent material is DNA, according to the position of DNA.

1. Cut down the gel which containing the target fragment and calculate the weight difference in the EP tube from adding the gel by weighing the tube before and after adding the gel.
2. Add Buffer B2 at a rate of 300-600 μL per 100 mg of agarose, depending on the weight and concentration of the gel.
3. Place the centrifuge tube in a 50°C water bath for 5-10 min and mix occasionally until the gel block is completely dissolved.
4. Transfer the entire dissolved solution to the adsorbent column and centrifuge at 8,000×g for 30 sec. Pour off the liquid from the collection tube and place the column in the same collection tube.
5. Add 500 μL Wash Solution to the column, centrifuge at 9,000×g for 30 sec and pour off the liquid from the collection tube and place the column in the same collection tube.
6. Repeat step 5 once.
7. Place the empty column and collection tube into a centrifuge and centrifuge at 9,000×g for 1 min. Add 15-40 μL of Elution Buffer to the center of the adsorbent membrane, leave at room temperature for 1-2 min, and centrifuge at 9,000×g for min. Store the resulting DNA solution at -20°C or use it for subsequent experiments.

Take 5mL of LB medium, add 5μL of Apramycin (working concentration 50μg/mL), add 100-200μL of pKC1139 strain and incubate in a shaker at 37℃, 220rpm overnight.

1. Bacterial solution was taken and centrifuged at 12000×g for 1 min, discard the supernatant.
2. Add 250 μL Buffer SP1 (store at 4℃, RNase A has been added) to suspension cell precipitation. (Add bacterial suspension and suspend bacteria)
3. Add 250 μL Buffer SP2, gently and fully turn up and down 4-6 times to mix evenly to fully lysis the bacteria, until a bright solution, this step should not exceed 5 min (lysis cells)
4. Add 350 μL Buffer SP3, gently and fully flip up and down 6-8 times to mix well, centrifuge at 12000×g for 1 min, discard filtrate (neutralization reaction, remove protein, genomic DNA and other impurities)
5. The supernatant was absorbed and transferred to the column (placed in a 2mL centrifuge tube), centrifuged at 12000×g for 1 min, and the filtrate was discarded
6. Add 500 μL Buffer W1, centrifuge at 12000×g for 1 min, discard the filtrate
7. Add 700 μL Buffer W2, centrifuge at 12000×g for 1 min, discard filtrate, wash with 700 μL Buffer W2 again in the same way, discard filtrate (clean, remove salt ions)
8. Put the column back into the 2 mL centrifuge tube and centrifuge at 12000×g for 1 min
9. The column was placed in a 1.5 mL centrifuge tube for 1-2 min. Eluent or deionized water was preheated at 65℃ and then added to the center of the column matrix, centrifuged at 12000×g for 1min. (Elution to obtain plasmid DNA)

A centrifuge tube containing Escherichia coli DH5a cells was placed on ice and mix with 10μL of recombinant product. Put it on ice for 30 minutes, 42°C water bath for 90 seconds, then immediately put on ice for 3 minutes. After that, 900μL of LB liquid medium was added into a centrifuge tube, then placed on a 37°C shaker and incubated at 200×g for 1 hour. Then, the cells were centrifuged at 5000×g for 5minutues, discarded the supernatant, and transferred the strain to an LB plate medium that contained Apramycin, and cultured overnight at 37°C.

1. PCR systems
   

   PCR systems
   Reagent	Volume（μL）
   2× Mix	10
   Primer F (10 μM)	1
   Primer R (10 μM)	1
   colony	0
   ddH2O	8
   Total	20

2. PCR program
   

   	PCR instrument program set-up.
   Steps	Temperature（℃）	Time
   1	98	3min
   2	98	30sec
   3	55	30sec
   4	72	20sec
   5	72	10min
   6	4	forever

   
   PS: Steps 2-4 cycle 30 times

1. Place the validated strain in a glycerol tube to obtain the target plasmid in E.coli.
2. Digest of plasmids with both EcoRI and HindIII.
   

   Reagent	Volume
   Plasmid pKC-M271_14685/M271_14690	20μL
   EcoRI	1μL
   HindIII	1μL
   CutSmart	5μL
   ddH2O	23μL
   Total	50μL

1. Inoculation: inoculate pKC-M271_14685/M271_14690 to 5 mL LB (plus 5 μL of Apramycin).
2. Extract pKC-M271_14685/M271_14690 recombinant plasmids
3. Conjugation transfer of recombinant plasmid into Streptomyces rapamycinicus.
   
   1. Use 1 μL of recombinant plasmid pKC-M271_14685/M271_14690 to transform Escherichia coli ET12567/pUZ8002 competent cells, coat LB solid plates with chloramphenicol, kanamycin, and Apramycin, and incubate overnight at 37°C. Inoculate colonies from the plates into 4mL LB liquid medium containing chloramphenicol, Kanamycin, and Apramycin, and incubate overnight at 37°C. Transfer 1 ml of bacterial broth to 50 ml of LB liquid medium containing chloramphenicol, Kanamycin, and Apramycin, and incubate until OD600 is 0.4-0.6. Centrifuge at 4000 rpm for 5 min and collect the bacteria. Wash the organisms twice with LB medium which has no antibiotics and resuspend the organisms in 500μL LB medium.
   2. Intensively spread 20 μL of Streptomyces rapae spore suspension on the oat medium and incubate at 30°C for 7-9 days. Collect fresh spore suspensions using 2 x YT liquid medium.
   3. Mix the E. coli suspension from step (1) and the spore suspension from step (2), coat onto M-ISP4 solid medium, and incubate at 30°C for 16 h. Coat with nalidixic acid and Apramycin and continue incubating at 30°C for 7-9 days.

1. Screening for single cross-over strains
   The strains grown in the previous step were scribed on the oat solid medium containing Apramycin and incubated at 30°C for 7-9 days until single colonies grew.
   The single colony was passaged twice on the oat solid medium containing Apramycin, and colonies were identified by PCR using the primers pKC1139-F and M14685-90-DDH-ID-R.
2. Filtrate double cross-over strains
   The Single cross-over strain was inoculated into 4 mL streptomyces seed medium and cultured at 30°C for 6 days. Transferred 40μL of the bacterial medium to 4ml fresh streptomyces seed medium, cultured at 30°C. Diluted the bacterial medium and coated it on the oat solid medium and cultured at 30°C for 7-9days. Cultured single colonies separately on the oat solid media containing Apramycin along with the solid media without antibiotics. Took only the colonies without antibiotics for PCR identification, the two primers used were M14685-90-SJH-ID-F and M14685-90-SJH-ID-R.