5. Measure the DNA concentration using a NanoDrop
spectrophotometer.
6. Set up the Gibson assembly reaction (20 μL) as in Table 3.
7. Incubate for 60 min at 50
C.
8. Add 80 μL of sterile MilliQ water to make a total volume of
100 μL.
9. Purify the reaction product using a MinElute PCR purification
kit, according to the manufacturer’s instructions. Carefully
elute the DNA with 10 μL of sterile MilliQ water.
10. Thaw 100 μL of Brevibacillus choshinensis electrocompetent
cells on ice (approximately 10 min), and mix the cells by flicking gently.
11. Transfer the cells to a chilled 1.5-mL centrifuge tube. Add
10 μL of the purified Gibson assembly reaction product.
12. Carefully transfer the cell/DNA mix into a chilled 0.2-cm
cuvette without introducing bubbles, and ensure that the
cells deposit across the bottom of the cuvette. Electroporate
using the following conditions for the Bio-Rad GenePulser
electroporator: 1.5 kV, 1000 Ω, and 25 μF. The typical time
constant is ~19 ms.
13. Immediately add 1 mL of pre-warmed MT medium to the
cuvette, gently mix by pipetting up and down twice, and then
transfer to the 17 mm  100 mm round-bottomed
culture tube.
14. Shake (200 rpm) at 37
C for 1 h.
15. Concentrate the cells via centrifugation as appropriate, and
then spread 100 μL of cells onto a pre-warmed MT/neomycin
agar plate.
Table 3
Gibson assembly reaction
Component
Size of DNA fragment
Amount per reaction
V L
360 bp
0.5 pmol
a
iRAT-MBP
1300 bp
0.5 pmol
a
V H
380 bp
0.5 pmol
a
VEC-NY
3400 bp
0.16 pmol
a
Gibson Assembly Master Mix (2Â)
--10 μL
Sterile MilliQ water
--To make 20 μL total volume
Total
20 μL
a
pmol ¼ (weight in ng)  1000/(base pairs  650 Da)
86
Norimichi Nomura et al.
spectrophotometer.
6. Set up the Gibson assembly reaction (20 μL) as in Table 3.
7. Incubate for 60 min at 50
C.
8. Add 80 μL of sterile MilliQ water to make a total volume of
100 μL.
9. Purify the reaction product using a MinElute PCR purification
kit, according to the manufacturer’s instructions. Carefully
elute the DNA with 10 μL of sterile MilliQ water.
10. Thaw 100 μL of Brevibacillus choshinensis electrocompetent
cells on ice (approximately 10 min), and mix the cells by flicking gently.
11. Transfer the cells to a chilled 1.5-mL centrifuge tube. Add
10 μL of the purified Gibson assembly reaction product.
12. Carefully transfer the cell/DNA mix into a chilled 0.2-cm
cuvette without introducing bubbles, and ensure that the
cells deposit across the bottom of the cuvette. Electroporate
using the following conditions for the Bio-Rad GenePulser
electroporator: 1.5 kV, 1000 Ω, and 25 μF. The typical time
constant is ~19 ms.
13. Immediately add 1 mL of pre-warmed MT medium to the
cuvette, gently mix by pipetting up and down twice, and then
transfer to the 17 mm  100 mm round-bottomed
culture tube.
14. Shake (200 rpm) at 37
C for 1 h.
15. Concentrate the cells via centrifugation as appropriate, and
then spread 100 μL of cells onto a pre-warmed MT/neomycin
agar plate.
Table 3
Gibson assembly reaction
Component
Size of DNA fragment
Amount per reaction
V L
360 bp
0.5 pmol
a
iRAT-MBP
1300 bp
0.5 pmol
a
V H
380 bp
0.5 pmol
a
VEC-NY
3400 bp
0.16 pmol
a
Gibson Assembly Master Mix (2Â)
--10 μL
Sterile MilliQ water
--To make 20 μL total volume
Total
20 μL
a
pmol ¼ (weight in ng)  1000/(base pairs  650 Da)
86
Norimichi Nomura et al.