Material&Methods

• 1.Parts
• 2.Primer list
• 3.Materials
• 3-1 Kit
• 3-2 Restriction Enzyme
• 3-3 Polymerase
• 3-4 DNA ligase
• 3-5 Marker
• 3-6 Organism
• 3-7 Antibiotics
• 3-8 Equipment
• 3-9 Backbone
• 3-10 Buffer
• 3-11 SDSPAGE&Westernblotting
• 3-12 B.xylophilus Experiment
• 4 Methods
  • 4-1 Miniprep
  • 4-2 Gel Extraction and PCR purification
  • 4-3 Restriction Enzyme Digestion
  • 4-4 Ligation
  • 4-5 Transformation
  • 4-6 PCR
  • 4-7 Sequencing
  • 4-8 RT-PCR
  • 4-9 RTqPCR
  • 4-10 Western blotting (Sample preparation of S. cerevisiae)
  • 4-11 Western blotting (Basic protocol)
  • 4-12 Culture using yeasts and measurement
  • 4-13 The way of taking photos
  • 4-14 Methods of taking movies
  • 4-15 Way of making agar pad
  • 4-16 Soaking

  1) Parts

  Basic Parts

  Composite Parts

  Primer List

  primer name	sequence	Length	Tm	GC%	Designer	Manufacturer
  tropomyosinF	GATCGAGAAGGACAACGCCC	20	65	60	Fukuda	Macrogen Japan Corp
  tropomyosinT7R	TAATACGACTCACTATAGGGCTTGTTTTCTCCGGC TTCGG	40	79	48	Fukuda	Macrogen Japan Corp
  tropomyosinT7F	TAATACGACTCACTATAGGGGATCGAGAAGGACAA CGCCC	40	79	50	Fukuda	Macrogen Japan Corp
  tropomyosinR	CTTGTTTTCTCCGGCTTCGG	20	66	55	Fukuda	Macrogen Japan Corp
  top-1F	GACTTTTCGTGCGTCTTCGG	20	64	55	Fukuda	Macrogen Japan Corp
  top-1T7R	TAATACGACTCACTATAGGGCCCGGTTGAAAAGCA AGTGT	40	78	45	Fukuda	Macrogen Japan Corp
  top-1T7F	TAATACGACTCACTATAGGGGACTTTTCGTGCGTC TTCGG	40	78	48	Fukuda	Macrogen Japan Corp
  top-1R	CCCGGTTGAAAAGCAAGTGT	20	64	50	Fukuda	Macrogen Japan Corp
  eef-1gF	GCAAAGGTTGCCGGTAAGAC	20	64	55	Fukuda	Macrogen Japan Corp
  eef-1gT7R	TAATACGACTCACTATAGGGGACGGCAGAGGCCAA CTTTA	40	78	48	Fukuda	Macrogen Japan Corp
  eef-1gT7F	TAA TACGACTCACTATAGGGGCAAAGGTTGCCGGT AAGAC	40	77	48	Fukuda	Macrogen Japan Corp
  eef-1gR	GACGGCAGAGGCCAACTTTA	20	64	55	Fukuda	Macrogen Japan Corp
  asbF	GGACTTTGGTCGTTGTCCCA	20	63	55	Fukuda	Macrogen Japan Corp
  asbT7R	TAATACGACTCACTATAGGGTCCACAGCGTCCTTC AAGTC	40	75	48	Fukuda	Macrogen Japan Corp
  asbT7F	TAATACGACTCACTATAGGGGGACTTTGGTCGTTG TCCCA	40	78	48	Fukuda	Macrogen Japan Corp
  asbR	TCCACAGCGTCCTTCAAGTC	20	61	55	Fukuda	Macrogen Japan Corp
  14-3-3proteinF-2	TGGAGGGTGATCTCGTCCAT	20	62	55	Fukuda	Macrogen Japan Corp
  14-3-3proteinT7R-2	TAATACGACTCACTATAGGG CAAGGGGGATGGTT GGTCTC	41	78	49	Fukuda	Macrogen Japan Corp
  14-3-3proteinT7F-2	TAATACGACTCACTATAGGG TGGAGGGTGATCTC GTCCAT	41	76	46	Fukuda	Macrogen Japan Corp
  14-3-3proteinR-2	CAAGGGGGATGGTTGGTCTC	20	65	60	Fukuda	Macrogen Japan Corp
  actinF	AATGGCTCCGGTATGTGCAA	20	65	50	Fukuda	Macrogen Japan Corp
  actinR	TGGTGGTGAAAGAGTAGCCG	20	62	55	Fukuda	Macrogen Japan Corp
  14-3-3zetaF	CCTACAAGAACGTGGTCGGT	20	61	55	Fukuda	Macrogen Japan Corp
  14-3-3zetaT7R	TAATACGACTCACTATAGGGTGTGAGTGCTCGCAA ACTGT	40	75	45	Fukuda	Macrogen Japan Corp
  14-3-3zetaT7F	TAATACGACTCACTATAGGGCCTACAAGAACGTGG TCGGT	40	76	48	Fukuda	Macrogen Japan Corp
  14-3-3zetaR	TGTGAGTGCTCGCAAACTGT	20	60	50	Fukuda	Macrogen Japan Corp
  14-3-3proF	TCTCGGTCGCCTACAAGAAC	20	61	55	Fukuda	Macrogen Japan Corp
  14-3-3proT7R	TAATACGACTCACTATAGGGCCAGTCTCTCCCGTC TCGTT	40	77	50	Fukuda	Macrogen Japan Corp
  14-3-3proT7F	TAATACGACTCACTATAGGGTCTCGGTCGCCTACA AGAAC	40	75	48	Fukuda	Macrogen Japan Corp
  14-3-3proR	CCAGTCTCTCCCGTCTCGTT	20	62	60	Fukuda	Macrogen Japan Corp
  AK1F	TCGGAGACGGTTGTGTGAAGATG	23	66	52	Yoshimoto	Macrogen Japan Corp
  AK1T7R	TAATACGACTCACTATAGGGCAAGCACGGGTTGA ATGGGT	41	79	46	Yoshimoto	Macrogen Japan Corp
  AK1T7F	TAATACGACTCACTATAGGGTCGGAGACGGTTGTG TGAAGATG	43	77	47	Yoshimoto	Macrogen Japan Corp
  AK1R	CAAGCACGGGTTGAATGGGT	20	66	55	Yoshimoto	Macrogen Japan Corp
  longAK2F	TCCGAATCATGGCGAGAACC	20	66	55	Yoshimoto	Macrogen Japan Corp
  longAK2T7R	TAATACGACTCACTATAGGGCATGCCGGTCAACGG ATAGT	40	78	48	Yoshimoto	Macrogen Japan Corp
  longAK2T7F	TAATACGACTCACTATAGGGTCCGAATCATGGCGA GAACC	40	78	48	Yoshimoto	Macrogen Japan Corp
  longAK2R	CATGCCGGTCAACGGATAGT	20	64	55	Yoshimoto	Macrogen Japan Corp
  shortAK2F	CCGTTTTGGATTTGTCGCGT	20	67	50	Yoshimoto	Macrogen Japan Corp
  shortAK2T7R	TAATACGACTCACTATAGGGTCGATCTTCTTGACC GTCGC	40	77	48	Yoshimoto	Macrogen Japan Corp
  shortAK2T7F	TAATACGACTCACTATAGGGCCGTTTTGGATTTGT CGCGT	40	79	45	Yoshimoto	Macrogen Japan Corp
  shortAK2R	TCGATCTTCTTGACCGTCGC	20	64	55	Yoshimoto	Macrogen Japan Corp
  gal1-1F	cccCGAATTCccccattatctt	22	68	50	Yoshimoto	Macrogen Japan Corp
  gal1-1R	CTCCCACACCAGCATCCAA	19	63	58	Yoshimoto	Macrogen Japan Corp
  gal1-2F	AAGCTGGGCGCCACTTT	17	64	59	Yoshimoto	Macrogen Japan Corp
  gal1-2R	CAAGTCGTTGCTGAAGAAACATTTCAC	27	66	41	Yoshimoto	Macrogen Japan Corp
  gal1-3F	ACTCGGCGTCCGGAG	15	61	73	Yoshimoto	Macrogen Japan Corp
  gal1-3R	tacccTGCAGgggagc	16	59	69	Yoshimoto	Macrogen Japan Corp
  gpd-1F	tctagaggatccccCGAATTC	21	63	52	Yoshimoto	Macrogen Japan Corp
  gpd-1R	CCGTTGACCGGCATGct	17	65	65	Yoshimoto	Macrogen Japan Corp
  gpd-2F	AATCCGCTGTGGCCGC	16	66	69	Yoshimoto	Macrogen Japan Corp
  gpd-2R	AACCACCTTCTTCGCACAGAAC	22	63	50	Yoshimoto	Macrogen Japan Corp
  gpd-3F	GACGTCCTTGGTGAAATGTTTC	22	61	45	Yoshimoto	Macrogen Japan Corp
  gpd-3R	gggaaaaccctggcgttac	19	64	58	Yoshimoto	Macrogen Japan Corp
  IK21	CCTCCACAAAGGCCTCTCCT	20	64	60	Yoshimoto	Macrogen Japan Corp
  IK22	CTTTATATTATCAATATTTGTGTTTGTGGAGGGGG	35	70	34	Yoshimoto	Macrogen Japan Corp
  IK23	ATTTATTGGAGAAAGATAACATATCATACTTTCCC	35	66	29	Yoshimoto	Macrogen Japan Corp
  IK24	AACCTTTATTGTGTGCACACTCAAAC	26	63	38	Yoshimoto	Macrogen Japan Corp
  IK25	GATAATATAAAGATGGGCAGCAGCCATCAT	30	69	40	Yoshimoto	Macrogen Japan Corp
  IK26	CCAATAAATCTATTCTTTAGCTCCTGACTCCAATA TTGTA	40	70	32	Yoshimoto	Macrogen Japan Corp
  IK27	ACTAGTGGATCCCCCCCTCCACAAAGGCCTCTCCT	35	81	60	Yoshimoto	Macrogen Japan Corp
  IK28	GAATTCCTGCAGCCCAACCTTTATTGTGTGCACAC TCAAAC	41	80	46	Yoshimoto	Macrogen Japan Corp
  IK60	GTGTAGGCCTCGGCATCCG	19	67	68	Yoshimoto	Macrogen Japan Corp
  IK61	TAATACGACTCACTATAGGGGTGGAGAGGGTGAAG GTGATG	41	77	49	Yoshimoto	Macrogen Japan Corp
  IK62	TAATACGACTCACTATAGGGTGCCATGTGTAATCC CAGCAG	41	77	46	Yoshimoto	Macrogen Japan Corp
  IK81	atgtatTCTAGAcTCCTCCACAAAGGCCTCTCCTG G	36	75	50	Dou	Macrogen Japan Corp
  IK82	GCCCCCTGCAtGCCCTCC	18	72	78	Dou	Macrogen Japan Corp
  IK83	GCGCAGGAGGGCaTGCAGG	19	73	74	Dou	Macrogen Japan Corp
  IK84	gtACTAGTgCTTTATATTATCAATATTTGTGTTTG TGGAGGGGGGGG	47	77	40	Dou	Macrogen Japan Corp
  IK85	GGGTCGCGGATCCgaaCtcATGG	23	75	65	Dou	Macrogen Japan Corp
  IK86	CGCTTCTTCCTGCCATgaGttcGGA	25	74	56	Dou	Macrogen Japan Corp
  IK87	atgtatTCTAGAcaatgggcagcagccatc	30	71	47	Dou	Macrogen Japan Corp
  IK88	gtACTAGTgCTATTCTTTAGCTCCTGACTCCA	32	66	44	Dou	Macrogen Japan Corp
  IK89	AAT CTA GAA GCG GCC GCA CAC GCT TT TTC	38	77	39	Dou	Macrogen Japan Corp
  IK90	GAACTAGTCAGAGATCTGAGTCCGGACTTGTACAG CTC	38	73	50	Dou	Macrogen Japan Corp
  IK91	ccgcaaaatcaggtgtgttg	20	63	50	Yoshimoto	Macrogen Japan Corp
  IK92	tatctttaccaattagtttcaatgtttagtaagg	34	63	26	Yoshimoto	Macrogen Japan Corp
  IK93	AATGACGCTGACGGTACAGG	20	61	55	Yoshimoto	Macrogen Japan Corp
  IK94	ATGCCCCAGACTGTGAGTTG	20	61	55	Yoshimoto	Macrogen Japan Corp
  IK95	ggttgtgtgaagatgaccgc	20	61	55	Yoshimoto	Macrogen Japan Corp
  IK96	tcttcagcagggacttgcag	20	62	55	Yoshimoto	Macrogen Japan Corp
  IK97	agcgttcaactagcagacca	20	59	50	Yoshimoto	Macrogen Japan Corp
  IK98	agggcagattgtgtggacag	20	61	55	Yoshimoto	Macrogen Japan Corp
  IK99	TGGCGAGAACCACCTTCTTC	20	63	55	Yoshimoto	Macrogen Japan Corp
  IK100	GCTCCCTGGTTGAACGTGTA	21	61	52	Yoshimoto	Macrogen Japan Corp
  IK21-2	CCTCCACAAAGGCCTCTCCT	20	64	60	Yoshimoto	Macrogen Japan Corp
  IK22-2	CTTTATATTATCAATATTTGTGTTTGTGGAGGGGG	35	70	34	Yoshimoto	Macrogen Japan Corp
  IK121	GCCTCTCCTGGGGTTTGAG	19	63	63	Ohara	Macrogen Japan Corp
  IK122	CTCCACAAACACAAATATTGATAATATAAAGatgg gcagc	40	73	35	Ohara	Macrogen Japan Corp
  IK129	CCTCCACAAACACAAATATTGATAATATAAAGatg ggcagcagccatcat	50	80	38	Ohara	Macrogen Japan Corp
  IK130	GGAAAGTATGATATGTTATCTTTCTCCAATAAATC TATTCTTTAGCTCCTGACTCCAATATTGTA	65	77	31	Ohara	Macrogen Japan Corp
  IK101	atgtcttctagagcggtaggt	21	56	48	Yoshimoto	Macrogen Japan Corp
  IK102	cctgattttgcggccgcatccgtcgaaactaagttctgg	39	85	54	Yoshimoto	Macrogen Japan Corp
  IK103	aacttagtttcgacggat gcggccgcaaaatcag g	36	83	50	Yoshimoto	Macrogen Japan Corp
  IK104	gcACTAGTtgaagcttctctatct	24	56	42	Yoshimoto	Macrogen Japan Corp
  IK105	atgtcttctagagccccattatcttagcctaaaaaaacc	39	73	38	Yoshimoto	Macrogen Japan Corp
  IK106	cctgattttgcggccgcctccatggtggcggc	32	90	69	Yoshimoto	Macrogen Japan Corp
  IK107	cccgccgccaccatggaggcggccgcaaaatcagg	35	94	71	Yoshimoto	Macrogen Japan Corp

  3) Materials

  Kit

  Name	Supplier
  Wizard® SV Gel and PCR	Promega
  FastGene™Plasmid Mini Kit	NIPPON Genetics Co.,Ltd

  Restriction Enzyme

  Name	Supplier
  EcoRI	TaKaRa, Promega
  PstI	TaKaRa, Promega
  SpeI	TaKaRa, Promega

  Polymerase

  Name	Supplier
  KAPA™HiFi HotStart ReadyMix (2x)	KAPABIOSYSTEMS
  KAPA2G™ Fast HotStart ReadyMix with dye (2x)	KAPABIOSYSTEMS
  KAPATaq™EXtra HotStart ReadyMix with dye	KAPABIOSYSTEMS

  DNA ligase

  Name	Supplier

  Marker

  Name	Supplier
  1kb DNA Ladder	TaKaRa

  Organism

  Name	Supplier
  E.coli DH5α Genotype	TaKaRa
  E.coli BL21(DE3)pLysS Competent Cells	Promega

  Antibiotics

  Name	Supplier

  Equipment

  Name	Supplier
  BioPhotometer	eppendorf
  LABO SHAKER	BIO CRAFT
  CO2 INCUBATOR	SANYO
  Pipette Controller	Biohit Midi Plus
  MiniCentrifuge Model GMC-060	LMS CO.,LTD.
  HIGH-SPEED REFRIGERATED CENTRIFUGE	TOMY
  HIGH-PRESSURE STEAM STERILIZER	TOMY
  VOTEX-GENE2	Scientific Industryes
  Scanning Electron Miniscope TM1000s	HITACHI
  Fluorescence Microscope BX61N-34-FL-1-D	OLYMPUS
  SCIECE IMAGING SYSTEM LAS-3000	Fuji film

  Backbone

  Name	Supplier
  pSB1C3	iGEM registry
  pSB1A2	iGEM registry
  pSB3C5	iGEM registry

  Buffer

  Name	Supplier
  Sodium Carbonate	Wako
  Sodium Bicarbonate	Wako
  Methanol(99.5%)	Wako
  Sodium Chloride	Wako
  SDS	nacalai tesque
  Trisaminomethane	nacalai tesque
  Potassium dihydrogenphosphste	SIGAMA-ALDRICH
  Potassium Chloride	Wako
  Glycine	Wako
  Agar, Powder	Wako
  Agarose XP	Wako
  10% Hydrochloric Acid	Wako

  SDSPAGE&Westernblotting

  IMMOBILON - P Blotting Sandwiches	Immobilon
  REAL GEL PLATE (10%)	BIO CRAFT
  BE-210(SDSPAGE)	BIO CRAFT
  BE-330	BIO CRAFT
  Amersham ECL Anti-Mouse IgG	GE healthcare
  Amersham ECL Anti-rabbit IgG	GE healthcare
  Amersham ECL Prime Blocking Reagent	GE healthcare
  Amersham ECL Prime WB Detection Reagent	GE healthcare

  B.xylophilus Experiment

  4) Methods

  Miniprep

  Minipreps were performed using FastGene™Plasmid Mini Kit and and Wizard® Plus SV Minipreps DNA Purification System according to the manufacturer's protocols.

  Gel Extraction and PCR purification

  Gel Extraction was performed using Wizard® SV Gel and PCR Clean-Up System according to the manufacturer's protocols.

  Restriction Enzyme Digestion

  Restriction enzyme treatment was performed using Takara Bio's or NEB's restriction enzymes according to the respective manufacturer's protocols.

  Ligation

  Ligation was performed using Takara Bio's Ligation High Ver.2 according to the manufacturer's

  Transformation

  1. Thaw competent cells on ice
  2. Add DNA sample (2.5μl) to competent cells. leave them on ice for 15 minutes
  3. Keep them in heating block for 45 seconds, then cool on ice for 2 minutes
  4. Add 90μl SOC liquid culture medium, then incubate for 45 minutes at 37°C
  5. Seed cells onto LB+antibiotics agar plate. Incubate for 24 hours at 37°C

  PCR

  PCR was performed using KAPA™HiFi HotStart ReadyMix (2x), KAPA™ 2G Robust HotStart ReadyMix (2x), PrimeSTAR® Max DNA Polymerase, GoTaq®, Takara Ex Taq, and Quick Taq® HS DyeMix according to the manufacturer's protocols

  Sequencing

  We outsourced the sequencing to Macrogen

  http://www.macrogen-japan.co.jp/cap_seq_0203.php

  RT-PCR

  We extracted B. Xylophilus whole mRNA using Sepazol®-RNA Ⅰ Super G according to the manufacturer's protocol.

  RT-PCR was performed using ~~~ according to the manufacturer's protocol.

  RTqPCR

  ????????

  Western blotting (Sample preparation of S. cerevisiae)

  1. Cultivate 1ml of S. cerevisiae culture whose OD600 values are 1.0
  2. Pour 1ml of the S. cerevisiae cell culture into 1.5ml tubes
  3. Centrifuge 5000rpm for 1min and remove the supernatant
  4. Resuspend with 30ul of SDS sample buffer
  5. Heat samples for 10min at 100°C using block incubater
  6. Vortex well

  Western blotting (Basic protocol)

  1. Apply sample to SDS-PAGE minigel (BIOCRAFT 15%)
  2. Soak the gel in transfer buffer
  3. Soak PVDF membrane in 100% methanol for 30 sec
  4. Soak PVDF membrane and filter paper in transfer buffer, leave for 5 min
  5. Set filter paper, membrane, gels, filter paper in this order to semidry transfer from anode to cathode
  6. Transfer the proteins from the gel to the membrane with 100 mA for 1 h
  7. Soak membrane in blocking buffer (dilution of 5 g ECL Blocking reagent by 100ml of TBST) for 1 h
  8. Wash for 5 min 3 times with TBST
  9. Apply 1' antibody (dilution of 10 μl of 1’antibody by 10ml of Blocking buffer) and shake for 1 h
  10. Wash for 5 min 3 times with TBST
  11. Apply 2' antibody (dilution of 4μl of 2’antibody by 10ml of Blocking buffer) and shake for 1 h
  12. Wash for 5 min 3 times with TBST
  13. Drain excess wash buffer from the washed membrane and place on flat surface, protein side up
  14. Add detection reagent onto the membrane, covering all of the membrane
  15. Incubate for 5 minutes at room temperature
  16. Drain off excess detection reagent by dabbing with Kimwipe
  17. Place the sample in the CCD camera compartment and record the images

  Culture using yeasts and measurement

  1. Prepare medium put on 3.5 cm plate.
  2. Drop liquid-cultured yeasts on the center of medium.
  3. Disperse them by bacteria spreader and dry for 30min to 1 hour. Or leave them as they are.
  4. Pipette suspension including nematodes on the medium.
  5. Culture it at 25 ℃

  The way of taking photos

  1. Culture yeasts expressing GFP over night and dilute them three times with DW or SD liquid medium.
  2. Drop 200-300μl of water on the medium after one or two days, and pipette it in order to spread over the medium.
  3. Suck out 12ul of water on the medium and drop it on microscope slide.
  4. Cover it with cover glass and observe whether nematodes feed yeasts expressing GFP by fluorescence microscope.

  Methods of taking movies

  1. Make agar pad.
  2. Extract nematodes from PDA medium, collect them into 1.5ml tube and centrifuge it.
  3. Remove supernatant and put 1.5ul of suspension with nematodes into gap of cover glasses.
  4. Snap microscope slide with the finger and reach suspension to the center of agarose medium.
  5. Observe whether nematodes feed yeast expressing GFP by microscope and take videos of it.

  Way of making agar pad

  1. Make medium of 2% agar and sterlize by autoclave.
  2. Put sterlized slide glass between two of slide glasses covered with "Kimwipes", drop agar medium, then rapidly cover agar medium with another slide glass.
  3. After the medium sodifies, reveal slowly covered slide glass.
  4. Pipette yeasts on the medium, cover the medium on cover glass, and culture it for 1-2days.

  Soaking

  1. Soak B. xylophilus in 50μl of 2μg /μl dsRNA solution.
  2. Shake nematode suspensions lightly (200rpm) for 4 hours at 20℃
  3. Wash nematodes in DDW several times and allow them to recover at 20℃ in distilled water.
  4. Observe and record their morphology, locomotion, and mortality at 4, 8, 12, 24, 36, 48, and 60 hours.
  5. Score juveniles/adults as being dead if they did not move.