Plasmid construction

(1)PCR to get thetarget DNA fragment

A target DNA fragment was obtained by PCR, and the PCR condition was showed in (Table 1 and Table 2 )

Table 1 The reaction system of PCR

Component / For 20 μL reation system / For 50 μL reation system
10×PCR buffer / 2.0 μL / 5.0 μL
dNTPs (2.5 mmol/L) / 1.5 μL / 4.0 μL
Primer-up (10 µmol/L) / 1.0 μL / 1.0 μL
Primer-down (10 µmol/L) / 1.0 μL / 1.0 μL
Template / 0.5 μL of plasmid or 1.0 μL of genome / 1 μL of plasmid or 1.5 μL of genome
rTaq / 0.5 μL / 1.0 μL
ddH2O / Add to 20 μL / Add to 50 μL

Table 2 The reaction program of PCR

Step / Procedure / Temperature / Time / Cycle
1 / Initial denaturation / 95℃ / 5 min / -
2 / Denaturation / 94℃ / 1 min / -
3 / Primer annealing / Uncertaintya / 1 min / -
4 / Extension / 72℃ / Uncertaintyb / -
5 / Go to step 2,3,4 / - / - / 25-30 cycles
6 / Eventually Extension / 72℃ / 10 min / -
7 / Storage ; / 4℃ / - / -

a: The primer annealing temperature is 5C below Tm of primers and no lower than 40C

b: The extension time is about 1 min/kb of expected product

(2) plasmid extraction

plasmid was extracted using a plasmid extraction kit (Omega, Madison, United States).

(3)Enzyme digestion reaction of the plasmid and target DNA fragment

All of the restriction enzymes used come from TaKaRa, Dalian, China. The enzyme digestion reaction was at 37 °C for 4 hours. The components of enzyme digestion system were showed in (Table 3).

Table 3 Components of enzyme digestion system

Components / Volume
10×Buffer / 10 μL
Plasmid or target DNA fragment / 40 μL
Restriction enzymes / 5.0 μL + 5.0 μL
ddH2O / Add to 100 μL

(4) Theconnection of plasmid and target DNA fragment

The ligase used come from TaKaRa, Dalian, China. The connection reaction was in a 16 °C water bath overnight. The connection system was showed in (Table 4).

Table 4 Connection system

Components / Volume
Solution I / 5.0 μL
Target DNA fragment / 4.0 μL or 3 μL
Plasmid / 1.0 μL or2 μL

(5) Make competent Escherichia coli strain DH5α cells

Competent E.coli DH5α cells were made using a competent cell creparation kit (TaKaRa, Dalian, China).

(6)Transformation DNA fragments into competent E. coli

① Remove competent cells (E.coli DH5a) from -80℃ freezer; thaw on wet ice about 5 minutes.

② Add recombinant DNA fragment to the competent cells by moving the pipette through the cells while dispensing. Gently tap tubes to mix.

③ Incubate on ice for 30 min.

④ Heat-shock cells 90 sec in a 42℃ water bath: do not shake.

⑤ Place cells on ice for 2 min rapidly.

⑥ Add 1 mL of LB medium to the tube containing cells, then incubate for 1 hours at 37°C with shaking speed around 100 rpm.

⑦Centrifuge at 13000 r/min for 1min and remove the supernatant.

⑧ Pipette 500 µL of sterile H2O into the transformation tube to re-suspend the pellet.

⑨ Plate 200 µL of the cell suspension onto the LB plate containing 100 mg/L Amp.

⑩ Incubate the plates overnight at 37 °C.

Transformants were screened on LB plate containing 100 mg/L Amp. Plasmid was extracted. Enzyme digestion reaction and electrophoresis were performed to identify the transformants.

2. Yeast transformation

Yeast transformation was carried out utilizing a lithium acetate procedure described previously [27].The detailedprocedure is describeas follow:

(1) Materials and Reagents

Deionized H2O

G418

PEG 3350

Lithium acetate dihydrate (LiAc)

Salmon sperm DNA

Transformation solution (see Recipes)

(2) Equipment

Water bath

Centrifuges

30°C shaker and incubator

Standard petri dishes

1.5 ml centrifuge tubes

(3) Recipes

Transformation solution: PEG 3350 [50% (w/v)] 240 µL
1 mol/L LiAc 36 µL
Salmon sperm DNA (2 g/L) 50 µL
Transforming DNA 34 µL
Total 360 µL

(4) Procedure

A: Make yeast competent cells

① Obtain yeast strains of interest and streak on YEPD plates. Let cells grow 2 d before inoculation.

② 1st Inoculation: Inoculate one colony into 5 mL YEPD liquid medium. Grow cells overnight at 30 °C with shaking speed around 180 rpm.

③ 2nd inoculation. Transfer 500 µL of the cells culture into 4.5 mL YEPD medium. Grow cells at 30 °C for 4 h.

④ Transfer 1 mL of the cells culture medium above into a new 1.5 mL sterile eppendorf tube and harvest cells by centrifugation at 10000 r/min for 1 min at 4 °C.

⑤ Wash cells twice with 1 mL water.

⑥ Wash cells with 1 mL of sterile 0.1mol/L LiAc buffer.

⑦ Re-suspend cells in 500 µL of sterile 0.1mol/L LiAc buffer.

⑧ Dispense 50 µL cells into a new 1.5 mL sterile eppendorf tube.

⑨ Centrifuge at 13000 r/min for 1 min at 4 °C to remove the supernatant.

B: Yeast cell transformation

① Make the transformation solution for the planned number of transformations plus one extra (negative control) (see the recipes).

② Add the solution to the cell pellet, vortex to re-suspend the cells.

③ Incubate in a 30°C stilling incubator.

④ Heat shock for 40 min in a 40°C water bath.

⑤ Centrifuge at 13000 r/min for 1 min and remove the supernatant.

⑥ Pipette 1 mL of liquid YEPD into the transformation tube to re-suspend the pellet, then grow cells for 4 hours at 30 °C with shaking speed around 100 rpm.

⑦ Centrifuge at 13,000 r/min for 1min and remove the supernatant.

⑧ Pipette 1 mL of sterile H2O into the transformation tube to re-suspend the pellet.

⑨ Plate 200 µL of the cell suspension onto the YEPD plate containing 300 mg/L G418.

⑩ Incubate plates at 30 °C for 2~4 d.

Transformants were screened on YEPD plate containing 300 mg/L G418. PCR was applied to verify the recombinant strains with accurate site integration.