Protocols & Notebook
Week 1 (7.10-7.16)
1. Metarhizium anisopliae resuscitation and subculturing:
1) Prepare solid PDA media (Solarbio, Beijing, China);
2) Make PDA plates;
3) Transfer M. anisopliae spores from the stock culture to new PDA plates using sterile pipette tips, three colonies per plate;
4) Culture at 30 ℃.
Notes & Results:
Succeeded. Fungi grew on new plates.
2. M. anisopliae resistance tests:
1) Prepare solid PDA media (Solarbio, Beijing, China);
2) Prepare G418 (100 mg/mL in water), glufosinate (100 mg/mL in
water), and benomyl (5 mg/mL in ethanol) stock solution, sterilize
using 0.22 µm filters (BKMAM, Changde, Hunan, China);
3) Prepare PDA plates containing 100 µg/mL, and 300 µg/mL G418;
4) Prepare PDA plates containing 100 µg/mL, and 300 µg/mL glufosinate;
5) Prepare PDA plates containing 10 µg/mL, 100 µg/mL, and 300 µg/mL benomyl;
6) Prepare PDA plates containing 0.2%, 0.6%, and 6% ethanol (control of benomyl group);
7) Transfer M. anisopliae spores from the stock culture to new plates using sterile pipette tips, two colonies per plate;
8) Culture at 30 ℃;
9) Measure the diameters of colonies on days 0, 3, 5, and 10.
Notes & Results:
M. anisopliae ACCC30104 was only susceptible to benomyl.
10 µg/mL benomyl was enough for screening.
3. M. anisopliae genomic DNA extraction:
1) Collect M. anisopliae hyphae from PDA plates using sterile pipette tips;
2) Use the GeneJet Genomic DNA Purification Kit (Thermo Fisher, Waltham, MA,
USA) to extract genomic DNA;
3) Measure the purity and concentration of the DNA samples with a
NanoDrop One spectrophotometer (Thermo Fisher, Waltham, MA,
USA).
Notes & Results:
Succeeded. Confirmed by Step 4.
4. PCR amplification of Pmcl1 and Pmcl1 (short):
1) 50 μL PCR: 19 μL deionized water, 25 μL Phusion High-Fidelity PCR
Master Mix (Thermo Fisher, Waltham, MA, USA), 2 μL forward primer
(10 μM), 2 μL reverse primer (10 μM), and 2 μL genomic DNA template;
2) The PCR program:
a) one cycle of 98°C for 30s;
b) 30 cycles of 98°C for 10s, 56°C for 30s, and 72°C for 2
min;
c) one cycle of 72°C for 5 min;
3) Run 1% gel electrophoresis to verify the size of amplified
fragments;
4) Extract DNA using the TIANgel Midi Purification Kit (Tiangen,
Beijing, China).
Notes & Results
:
Succeeded.
Week 2 (7.17-7.23)
5. Recombinant vector construction:
1) Digest pBARGPE1-EGFP-BenA with FastDigest NheI and EcoRI (Thermo
Fisher, Waltham, MA, USA);
2) PCR amplification of SuperNova (suicide gene), KillerRed (suicide
gene), and LqhIT2 (toxin) (all synthesized by GENEWIZ, Suzhou, Jiangsu, China);
3) Run 1% gel electrophoresis to verify the size of amplified
fragments;
4) Extract DNA using the TIANgel Midi Purification Kit (Tiangen,
Beijing, China);
5) Measure the purity and concentration of the DNA samples with a
NanoDrop One spectrophotometer (Thermo Fisher, Waltham, MA,
USA);
6) Use Vazyme ClonExpress Ultra One Step Cloning Kit (Vazyme,
Nanjing, Jiangsu, China):
10 μL reaction system: 5 μL 2× Mix, 3 μL digested plasmid, 1 μL
Pmcl1/Pmcl1 (short), and 1 μL SuperNova/KillerRed/LqhIT2;
7) Recombinant vectors:
a) pBARGPE1-Pmcl1-SuperNova;
b) pBARGPE1-Pmcl1(short)-SuperNova;
c) pBARGPE1-Pmcl1-KillerRed;
d) pBARGPE1-Pmcl1(short)-KillerRed;
e) pBARGPE1-Pmcl1(short)-LqhIT2;
5) Transform the recombinant vectors into Escherichia coli DH5α;
6) Screen on LB plates containing 100 μg/mL;
7) Culture the positive transformants in liquid LB;
8) Extract plasmids using the TIANprep Rapid Mini Plasmid Kit
(Tiangen, Beijing, China);
9) Confirm by DNA sequencing (GENEWIZ, Suzhou, Jiangsu,
China).
Notes & Results:
Succeeded on the first try:
pBARGPE1-Pmcl1(short)-SuperNova, pBARGPE1-Pmcl1(short)-KillerRed, and
pBARGPE1-Pmcl1(short)-LqhIT2.
Failed once: pBARGPE1-Pmcl1-SuperNova, pBARGPE1-Pmcl1-KillerRed.
Succeeded after adjusting the proportion of Gibson Assembly to 5
μL 2× Mix, 2.5 μL digested plasmid, 1.5 μL Pmcl1, and 1 μL
SuperNova/KillerRed/LqhIT2
Week 3 (7.24-7.30)-Week 4 (7.31-8.6)
Protoplast transformation of M. anisopliae:
1) Prepare 80 mL liquid PDB media (Solarbio, Beijing, China);
2) Culture M. anisopliae spores in PDB at 30 ℃, 180rpm for 24h;
3) Filter with filter paper to collect mycelia;
4) Prepare 20 mL lysis buffer: 1% cellulase, 2% snailase, and 0.8M
sorbitol in phosphate buffer, pH 5.8;
5) Incubate mycelia in lysis buffer at 30 ℃, 80 rpm for 4h;
6) Filter with sterile cotton to remove the remaining mycelia;
7) Confirm the concentration of protoplasts using a hemocytometer
under a Murzider MSD-S280 light microscope (Murzider, Dongguan,
Guangdong, China);
8) Centrifuge at 600 ×g for 5 min, and discard the
supernatant;
9) Prepare 20 mL STC buffer: 20% sucrose (w/v), 10mM Tris-HCl, and
50mM CaCl2 in water, pH8.0;
10) Resuspend the protoplasts in STC buffer and adjust the concentration to 2 × 107 protoplasts/mL;
11) Add 15 μg recombinant vector to 200 μL protoplast
suspension;
12) Sit at room temperature for 20 min;
13) Prepare 10 mL PTC buffer: 40% PEG4000 (w/v) in STC buffer;
14) Add 1.25 mL PTC buffer to the protoplast suspension from Step 11,
and invert several times to obtain a homogeneous mixture;
15) Sit at room temperature for 20 min;
16) Prepare 50 mL liquid TB3 media: 3% Yeast Extract (w/v), 3%
Casamino Acids (w/v), and 20% sucrose (w/v) in water;
17) Add 5 mL TB3 culture containing 100 μg/mL ampicillin to the
mixture from Step 15;
18) Culture overnight at 30 ℃, 180 rpm;
19) Centrifuge the mixture at 3000 ×g for 5 min, and discard the
supernatant;
20) Resuspend in 200 μL STC buffer;
21) Prepare 200 mL solid TB3 media: 3% Yeast Extract (w/v), 3% Casamino Acids (w/v), 20% sucrose
(w/v), and 0.7 % low melting point agarose in water;
22) Add 10 ml TB3 media containing 10 μg/mL benomyl and 100 μg/mL
ampicillin to the mixture from Step 20, and gently rotate to mix thoroughly;
23) Pour 10 mL mixture into a 9 cm Petri dish as the bottom culture
media;
24) Incubate overnight at 30 ℃;
25) The next day, pour 10 ml solid TB3 media containing 10 μg/mL
benomyl above the bottom culture media;
26) Incubate for 3 days at 30 °C;
27) Pick a single colony and subculture on the PDA plates containing
10 μg/mL benomyl;
28) Subculture twice to ensure stable transformants;
29) Perform colony PCR to ensure positive transformants.
Notes & Results:
Cellulase was only partially soluble in water. So, after
filtration, its concentration would be less than 1%, and it also
worked.
We were not able to get enough hyphae after step 2) on the first
try, so we elongated this step to 40 hours.
We were not able to see colonies after step 26). It took two more
days for the colonies to appear.
Week 5 (8.7-8.13)-Week 6 (8.14-8.18)
6. Virulence tests of engineered M. anisopliae:
1) Purchase larvae of the greater wax moth (Galleria mellonella) from Keyun Bio Co., Ltd., Shanghai, China;
2) Add 1 mL sterile water to each M. anisopliae plate, and gently scrape the spores off the agar using a cell
spreader;
3) Filter with sterile cotton to remove the mycelia;
4) Confirm the concentration of spores using a hemocytometer under a
Murzider MSD-S280 light microscope (Murzider, Dongguan, Guangdong,
China);
5) Centrifuge at 3000 ×g for 5 min, and discard the
supernatant;
6) Resuspend the spores in sterile water and adjust the concentration to 1 × 107, 1 × 106, 1 × 105 spores/mL;
7) Place a moist tissue paper in each Petri dish to maintain the
humidity, and place five larvae in each Petri dish;
8) Divide larvae into ten groups (15 larvae/group) based on
different treatments:
a) Group I: uninoculated control larvae;
b) Group II: inoculate larvae with 1 × 107 original M. anisopliae spores;
c) Group III: inoculate larvae with 1 × 106 original M. anisopliae spores;
d) Group IV: inoculate larvae with 1 × 105 original M. anisopliae spores;
e) Group V: inoculate larvae with 1 × 107 M. anisopliae spores expressing LqhIT2;
f) Group VI: inoculate larvae with 1 × 106 M. anisopliae spores expressing LqhIT2;
g) Group VII: inoculate larvae with 1 × 105 M. anisopliae spores expressing LqhIT2;
h) Group VIII: inoculate larvae with 1 × 107 M. anisopliae spores expressing Pmcl1(short)-SuperNova;
i) Group IX: inoculate larvae with 1 × 106 M. anisopliae spores expressing Pmcl1(short)-SuperNova;
j) Group X: inoculate larvae with 1 × 105 M. anisopliae spores expressing Pmcl1(short)-SuperNova;
9) Place the Petri dishes in a constant temperature incubator at 30 °C;
10) Record the death numbers of each group every morning, and remove the
corpses.
Notes & Results:
Water needed to be added every other day to maintain the
humidity.
If corpses turned soft and black in two days after death, it was
very likely that they were colonized by bacteria. Bacteria would
outcompete the fungi and the corpses cannot be used in further
assays.
Results showed that LqhIT2 strains killed pests faster than WT.
Week 6 (8.14-8.18)
7. Spore formation test of engineered M. anisopliae:
1) Add three more groups (15 larvae/group) based on different
treatments:
k) Group XI: inoculate larvae with 1 × 107 M. anisopliae spores expressing Pmcl1-SuperNova;
l) Group XII: inoculate larvae with 1 × 107 M. anisopliae spores expressing Pmcl1(short)-KillerRed;
m) Group XIII: inoculate larvae with 1 × 107 M. anisopliae spores expressing Pmcl1-KillerRed;
2) Collect the dead larvae on day 5 from Groups II, V, VIII, XI,
XII, and XIII;
3) Place the Petri dishes under sunshine until spores form outside
the insect body;
4) Place the corpses in a 1.5 mL centrifuge tube, add 500 μL water, and vortex for 2 min to collect the spores;
5) Confirm the concentration of spores using a hemocytometer under a
Murzider MSD-S280 light microscope (Murzider, Dongguan, Guangdong,
China).
Notes & Results:
The suspension should be filtered after step 4) to remove the
hyphae.
Results showed that Group VIII produced the least spores.
8. Spore germination rate test of engineered M. anisopliae:
1) Centrifuge the spore suspensions from Step 8 at 3000 ×g for 5 min, and discard the supernatant;
2) Resuspend in liquid PDB media, and adjust the concentration to 5
× 105 spores/mL;
3) Incubate at 30 °C, 180 rpm for 10h (6h on day 1, store at 4 °C
overnight, 4h on day 2);
4) Observe under a Murzider MSD-S280 light microscope (Murzider,
Dongguan, Guangdong, China) to determine the germination rate.
Notes & Results:
We checked the germination rate of WT spores every 2 hours to
find an appropriate time point for the germination rate assay. It
turned out that 10 hours was a good time point.
Results showed that all the groups had a similar germination
rate.
