Team:Kyoto/Materials&Methods

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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 SDS-PAGE&Westernblotting
• 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 Synthesis of dsRNA
• 4-17 Soaking

1) Parts

Basic Parts

Composite Parts

2) Primer List

primer name	sequence	Length	Tm	GC%	Designer	Manufacturer
tropomyosinF	GATCGAGAAGGACAACGCCC	20	65	60	Fukuda	Macrogen Japan Corp
tropomyosinT7R	TAATACGACTCACTATAGGGCTTGTTTTCTCCGGCTTCGG	40	79	48	Fukuda	Macrogen Japan Corp
tropomyosinT7F	TAATACGACTCACTATAGGGGATCGAGAAGGACAACGCCC	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	TAATACGACTCACTATAGGGCCCGGTTGAAAAGCAAGTGT	40	78	45	Fukuda	Macrogen Japan Corp
top-1T7F	TAATACGACTCACTATAGGGGACTTTTCGTGCGTCTTCGG	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	TAATACGACTCACTATAGGGGACGGCAGAGGCCAACTTTA	40	78	48	Fukuda	Macrogen Japan Corp
eef-1gT7F	TAA TACGACTCACTATAGGGGCAAAGGTTGCCGGTAAGAC	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	TAATACGACTCACTATAGGGTCCACAGCGTCCTTCAAGTC	40	75	48	Fukuda	Macrogen Japan Corp
asbT7F	TAATACGACTCACTATAGGGGGACTTTGGTCGTTGTCCCA	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 CAAGGGGGATGGTTGGTCTC	41	78	49	Fukuda	Macrogen Japan Corp
14-3-3proteinT7F-2	TAATACGACTCACTATAGGG TGGAGGGTGATCTCGTCCAT	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	TAATACGACTCACTATAGGGTGTGAGTGCTCGCAAACTGT	40	75	45	Fukuda	Macrogen Japan Corp
14-3-3zetaT7F	TAATACGACTCACTATAGGGCCTACAAGAACGTGGTCGGT	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	TAATACGACTCACTATAGGGCCAGTCTCTCCCGTCTCGTT	40	77	50	Fukuda	Macrogen Japan Corp
14-3-3proT7F	TAATACGACTCACTATAGGGTCTCGGTCGCCTACAAGAAC	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	TAATACGACTCACTATAGGGCAAGCACGGGTTGAATGGGT	41	79	46	Yoshimoto	Macrogen Japan Corp
AK1T7F	TAATACGACTCACTATAGGGTCGGAGACGGTTGTGTGAAGATG	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	TAATACGACTCACTATAGGGCATGCCGGTCAACGGATAGT	40	78	48	Yoshimoto	Macrogen Japan Corp
longAK2T7F	TAATACGACTCACTATAGGGTCCGAATCATGGCGAGAACC	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	TAATACGACTCACTATAGGGTCGATCTTCTTGACCGTCGC	40	77	48	Yoshimoto	Macrogen Japan Corp
shortAK2T7F	TAATACGACTCACTATAGGGCCGTTTTGGATTTGTCGCGT	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	CCAATAAATCTATTCTTTAGCTCCTGACTCCAATATTGTA	40	70	32	Yoshimoto	Macrogen Japan Corp
IK27	ACTAGTGGATCCCCCCCTCCACAAAGGCCTCTCCT	35	81	60	Yoshimoto	Macrogen Japan Corp
IK28	GAATTCCTGCAGCCCAACCTTTATTGTGTGCACACTCAAAC	41	80	46	Yoshimoto	Macrogen Japan Corp
IK60	GTGTAGGCCTCGGCATCCG	19	67	68	Yoshimoto	Macrogen Japan Corp
IK61	TAATACGACTCACTATAGGGGTGGAGAGGGTGAAGGTGATG	41	77	49	Yoshimoto	Macrogen Japan Corp
IK62	TAATACGACTCACTATAGGGTGCCATGTGTAATCCCAGCAG	41	77	46	Yoshimoto	Macrogen Japan Corp
IK81	atgtatTCTAGAcTCCTCCACAAAGGCCTCTCCTGG	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	gtACTAGTgCTTTATATTATCAATATTTGTGTTTGTGGAGGGGGGGG	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	GAACTAGTCAGAGATCTGAGTCCGGACTTGTACAGCTC	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	CTCCACAAACACAAATATTGATAATATAAAGatgggcagc	40	73	35	Ohara	Macrogen Japan Corp
IK129	CCTCCACAAACACAAATATTGATAATATAAAGatgggcagcagccatcat	50	80	38	Ohara	Macrogen Japan Corp
IK130	GGAAAGTATGATATGTTATCTTTCTCCAATAAATCTATTCTTTAGCTCCTGACTCCAATATTGTA	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

3-1 Kit

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

3-2 Restriction Enzyme

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

3-3 Polymerase

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

3-4 DNA ligase

Name	Supplier
T4 DNA ligase	TaKaRa

3-5 Marker

Name	Supplier
1kb DNA Ladder	TaKaRa

3-6 Organism

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

3-7 Antibiotics

Name	Supplier
Chloramphenicol	Wako
Ampicillin	Wako

3-8 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

3-9 Backbone

Name	Supplier
pSB1C3	iGEM registry
pSB1A2	iGEM registry
pSB3C5	iGEM registry

3-10 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

3-11 SDS-PAGE&Westernblotting

IMMOBILON - P Blotting Sandwiches	Immobilon
REAL GEL PLATE (10%)	BIO CRAFT
BE-210(SDS-PAGE)	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

4) Methods

4-1 Miniprep

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

4-2 Gel Extraction and PCR purification

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

4-3 Restriction Enzyme Digestion

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

4-4 Ligation

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

4-5 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

4-6 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

4-7 Sequencing

We outsourced the sequencing to Macrogen

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

4-8 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.

4-9 qRT-PCR

We inserted 3 plasmids written on the lower left into 2 types of yeasts written on the lower right.

p14 Gal1p-AK1 p26 GPDp-AK1 p100 GPF-GFP

MKY13 WT MKY117 ski2Δ

Regarding p14, we conducted

1. Create culture with raffinose medium as primary culture.
2. Dilute it in glucose medium and galactose medium and incubate for 12-15 hours.
3. Collect at log phase of OD = 1.1- 1.8 (MKY13) 0.6 - 1.2 .(MKY117)
4. Collect RNA by Lucigen's MasterPure Yeast RNA purification kit. (http://www.lucigen.com/home.php?cat=273)
5. Do DNase treatment according to the manual for 20 minutes.
6. Dissolve the obtained total RNA in 15 μL and dilute it to prepare a standard curve sample (using p14 sample and p100 sample). The measurement sample was diluted 5000 times with water and used. Analysis is ABI Step-one plus. SuperScript III Platinum SYBR qRT-PCR kit (https://www.thermofisher.com/order/catalog/product/11732020) was used.

The three following primers were used :

• IK91-IK92: This amplifies the loop region of hairpin RNA
• IK95-IK96: This amplifies the inside of AK1 mRNA
• Kota 030 - Kota 031: It amplifies near the 5 'end of 25S rRNA. (Fujii Kitabatake et al., 2009)

Quantitation was performed with n = 3 biological triplicate, and the variation in RNA recovery amount was standardized by dividing the quantified value of loop and the quantified value of AK1 by the quantitative value of 25S rRNA, respectively.

4-10 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

4-11 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

4-12 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 ℃

4-13 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.

4-14 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.

4-15 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.

4-16 Synthesis of dsRNA

We synthesyzed dsRNA by "MEGAscript™ T7 Transcription Kit."
https://www.thermofisher.com/order/catalog/product/AM1334

4-17 Soaking

We followed this paper as for soaking.
https://link.springer.com/content/pdf/10.1007%2Fs10658-012-0035-0.pdf