Bacmid preparation
The detail of silkworm expression system and bacmid preparation are described in previous reports9,10.
Day 1
- Add 100 – 1000 ng of pFastBac HTb L-OPA1 into 100 μl of BmDH10bac competent ;cells. Note that BmDH10bac is needed for silkworm expression, not BmDH10.
- Heat shock at 42 °C for 45 sec and place on the ice for 2 min.
- Add 1.4 ml LB medium and incubate at 37 °C for 1 h in shaking incubator.
- Add tetracycline to a final concentration of 10 μg/ml and incubate at 37 °C overnight.
Day 2
- After overnight incubation, add gentamicin to a final concentration of 7 μg/ml and incubate for 2 h at 37 °C.
- Plate preparation: 20 μl of 100 mM IPTG and 70 μl of 2% Bluo-Gal were spread on LB plate containing kanamycin (final concentration of 50 μg/ml) and gentamycin (final concentration of 7 μg/ml).
- The cultured bacteria in step 5 was spread 150 μl on the LB plate prepared in step 6 and incubate for 15 h at 37 °C.
Day 3
- Pick up white colonies from the plate in step 7, then confirm correct transposition by colony PCR using following universal primers.
M13 forward (-40) 5′-GTTTTCCCAGTCACGAC-3′
M13 Reverse 5′-CAGGAAACAGCTATGAC-3′
In the case of L-OPA1, if transposition has occurred correctly, you can see the band at 5000 bp.
- Select the colony with correct transposition, and amplified in 3 ml LB-Kanamycin (final concentration of 50 μg/ml) medium.
Day 4
- Prepare glycerol stocks; mix aliquots (750 μl) of the saturated culture with 150 μl of 60% glycerol, then freeze in liquid nitrogen and store at -80 °C
For the large scale culture; Pour 1.5 ml of amplified cell into 100 ml LB- Kanamycin (final concentration of 50 μg/ml) medium and incubate overnight at 37 °C in shaking incubator.
Day 5
- Harvest the cultured cells by centrifugation at 4000 xg for 10 min at 4 °C. If you hope bacmid DNA preparation later, freeze the cell pellet and stored at freezer.
- Purified Bacmid DNA with a QIAfilter plasmid kit. We use Qiafilter plasmid Midi for 50 ml cultured cell.
Resolve the air-dried bacmid DNA pellet with 80 μl ultrapure water and determine the concentration. In our case, more than 160 μg of bacmid DNA is recovered from 50 ml culture.
L-OPA1 expression in silkworm fat body
Day1
- Prepare bacmid DNA-liposome mix:
Mix with 1.5μg bacmid DNA with 3 μl DMRIE-C per each larva. After incubation for 45 min at room temperature, add 50 μl ultrapure water.
We usually treat/rear 60 silkworm larvae at the same time.
(Mix 1.5 μg x 60 = 90 μg bacmid with 3 μl x 60 = 180 μl of DMRIE-C, after incubation add 50 μl x 60 = 3000 μl ultrapure water.)
- Inject 50 μl mixture directly into the dorsal side of a silkworm larva with 30-gauge needle.
- After 30 min, start feed artificial diet Silkmate 2S.
- Culture silkworm for 6 days in climate chamber under 25 °C, 80% humidity.
We usually rear 15 silkworm larvae in 22 x 16 x 6.5 cm box with breathing holes and feed silkmate 2S everyday in the morning as bellow.
For 15 silkworm larvea
day 1 (20 g), day 2 (30 g), day 3 (45 g,) day 4 (50 g), day 5 (50 g), day 6 (35 g).
Before feeding, discard feces.
Note that silkworm larva only eat fresh mulberry leaves or artificial diet like as silkmate 2S.
Day 7
Isolation of fat bodies from 15 silkworm larvae
- Prepare 40 ml of PB buffer.
- Isolate infected fat bodies by manually and collect ice-cold PB buffer (see Fig 1b in associated publication).
- Homogenize collected fat bodies in a 50 ml glass-teflon potter homogenizer with 10 strokes at 1300 rpm and transfer to new 50 ml conical tube.
Freeze in liquid nitrogen and store at -80 °C.
L-OPA1 purification from silkworm fat body
Day 1
Purification of L-OPA1 from fat bodies isolated from 60 silkworm larvae.
- Prepare 200 ml of PB buffer containing 10 mM imidazole and 1% dodecyl maltoside (DDM).
As described above, the fat bodies isolated from 15 silkworms were stored in one tube, thus in following procedure (step 2-10), handle 4 tubes at once except for sonication.
- Thaw the frozen fat bodies on the ice and transfer to 50 ml glass beaker.
- Sonicate fat bodies (Cycle time 2, Duty cycle 50, Output control 9. for 10 min) on the ice and transfer to new 50 ml conical tube.
- Centrifuge at 500 xg for 5 min at 4 °C with swing rotor (Beckman JS4.2).
- Remove top floating and decant supernatant to new 50 ml conical tube gently.
- Centrifuge supernatant at 14,000 xg for 60 min at 4 °C with angle rotor (Beckman JA12).
- Remove supernatant.
- Suspend pellet with 40 ml of ice-cold PB containing 10 mM imidazole and 1% DDM and incubate in 50 ml conical tube for 120 min at 4 °C with gentle agitation.
- Centrifuge at 14,000 xg for 30 min at 4 °C with angle rotor (Beckman JA12).
- Collect supernatant in new 50 ml conical tube and incubate with 750 μl (bed volume) of Ni-Sepharose 6 fast flow beads overnight at 4 °C with gentle agitation
Day 2
Purification of L-OPA1 from fat bodies isolated from 60 silkworm larvae
- Prepare following buffer
16 ml of wash 1 buffer (RB containing 10 mM imidazole and 0.1% DDM)
16 ml of wash 2 buffer (RB containing 20 mM imidazole and 0.1% DDM)
12 ml of elute buffer (RB containing 250 mM imidazole and 0.1% DDM)
- Centrifuge at 50 xg for 1 min at 4 °C with swing rotor (Beckman JS4.2).
- Remove supernatant and wash Ni-Sepharose 6 with 1 ml of ice-cold PB containing 10 mM imidazole and 1% DDM.
- Repeat step 12-13.
- Suspend Ni-Sepharose 6 with ice-cold PB containing 10 mM imidazole and 1% DDM
- Load Ni- Sepharose 6 into Econo-column carefully from 4 of 50 ml conical tubes.
- Wash with 16 ml of ice-cold wash 1 buffer.
- Wash with 16 ml of ice-cold wash 2 buffer.
- Elute 8 times with 1.5 ml of ice-cold elute buffer.
- Analyze each fraction with SDS-PAGE and measure the concentration with Bradford assay.
In our case, L-OPA1 major band appears in Fraction 2-3 and total yield is more than 2 mg from 60 silkworm larvae.
Freeze in liquid nitrogen and store at -80 °C.
Detergent exchange
- Prepare following buffer.
50 ml of RB containing 0.1% DDM
20 ml of wash (Mega8) buffer (RB containing 10 mM imidazole and 2.5% Mega-8)
5 ml of elute (Mega8) buffer (RB containing 250 mM imidazole and 2.5% Mega-8)
- Dilute the eluted fraction (with L-OPA1) 10-fold with RB containing 0.1% DDM.
- Allow the diluted protein into Econo-column with 1 ml (bed volume) Ni-Sepharose 6 by gravity flow at 4 °C.
- Wash with 5 ml of RB containing 0.1% DDM.
- Wash with 20 ml of wash (Mega8) buffer.
- Elute 8 times with 0.5 ml of ice-cold elute (Mega8) buffer.
- Analyze each fraction with SDS-PAGE and measure the concentration with Bradford assay.
- Freeze in liquid nitrogen and store at -80 °C.