Contents

• 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 Backbones
  • 3-10 Buffer
  • 3-11 SDSPAGE/Western blotting
  • 3-12 Fluorescence Microscope
• 4 Methods
  • 4-1 Miniprep
  • 4-2 Gel Extraction
  • 4-3 Restriction Enzyme Digestion
  • 4-4 Ligation
  • 4-5 Transformation
  • 4-6 PCR
  • 4-7 Sequencing
  • 4-8 RT-PCR
  • 4-9 Western blotting (basic protocol)
  • 4-10 Western blotting (anti-NoVLP)
  • 4-11 Western blotting (Membrane fraction)
  • 4-12 Western blotting (Whole cell)
  • 4-13 Growth Curve Drawing
  • 4-14 Cellulose Affinity Assay with spectrophotometer
  • 4-15 Cellulose Affinity Assay with Fluorescence Microscope
  • 4-16 Scanning Electron Microscopy (2015 Summer Experiment)
  • 4-17 Scanning Electron Microscopy (2016 Summer Experiment)

  Parts

  Basic Parts

  Composite Parts

  Primer list

  Primer name	Sequence	Length	Tm	GC%	Designer	Manufacturer
  m.scfv Fw	TGTTTCTCCAGATGAACAGCCTGAGAG	27bp	66	47	Li	Thermo Fisher Scientific Inc.
  m.scfv Rv	TCATCTGGAGAAACAACACATACTTGG	27bp	67	44	Li	Thermo Fisher Scientific Inc.
  c.scfv Fw	AGGCGGATCCGGTATGGCCGAGGTGCAGC	29bp	74	69	Li	Thermo Fisher Scientific Inc.
  c.scfv Rv	TACTAGTAGCGGCCGCTGCAGTACACCTAGGACGGT GACCTTGG	44bp	75	60	Li	Thermo Fisher Scientific Inc.
  m.BAN Fw	TGCCGACCATGGCCGAGGTGCAGCTG	26bp	72	69	Li	Thermo Fisher Scientific Inc.
  m.BAN RV	TCGGCCATGGTCGGCAGGGTGAAC	24bp	69	67	Li	Thermo Fisher Scientific Inc.
  VF2	TGCCACCTGACGTCTAAGAA	20bp	57	50	iGEM	Thermo Fisher Scientific Inc.
  VR	ATTACCGCCTTTGAGTGAGC	20bp	56	50	iGEM	Thermo Fisher Scientific Inc.
  BAN+scFv.Fw	ACTAGAGAAAGAGGAGAAATACTAGATGGCGTTC	34bp	62	41	Uchino	Macrogen Japan Corp.
  J23114+RBS.Rv	GAACGCCATCTAGTATTTCTCCTCTTTCTCTAGTGC TAGCATTGTACCTAGGACTGAGCTAGC	63bp	72	46	Uchino	Macrogen Japan Corp.
  J23108+RBS.Rv	GAACGCCATCTAGTATTTCTCCTCTTTCTCTAGT	34bp	62	41	Uchino	Macrogen Japan Corp.
  J23100+RBS.Rv	GAACGCCATCTAGTATTTCTCCTCTTTCTCTAGTGC TAGCACTGTACCTAGGACTGAGC	59bp	72	47	Uchino	Macrogen Japan Corp.
  Cex.Fw	GGTCCGGCCGGGTGCCAGGTG	21bp	71	81	Uchino	Macrogen Japan Corp.
  Clos.Fw	TCATCAATGTCAGTTGAATTTTACAACTCTAAC	33bp	58	30	Uchino	Macrogen Japan Corp.
  INP_His_Cex.Rv	CACCTGGCACCCGGCCGGACCATGATGATGATGATG ATGGGATCCGCCTCCTGCA	55bp	80	62	Uchino	Macrogen Japan Corp.
  INP_His_Clos.Rv	GTTAGAGTTGTAAAATTCAACTGACATTGATGAATG ATGATGATGATGATGGGATCCGCCTCCTGCA	67bp	72	40	Uchino	Macrogen Japan Corp.
  BAN_His_Cex.Rv	CACCTGGCACCCGGCCGGACCATGATGATGATGATG ATGGGTCGGCAGGGTGAAC	55bp	80	62	Uchino	Macrogen Japan Corp.
  BAN_His_Clos.Rv	GTTAGAGTTGTAAAATTCAACTGACATTGATGAATG ATGATGATGATGATGGGTCGGCAGGGTGAAC	67bp	72	40	Uchino	Macrogen Japan Corp.
  scFv_ctl_Fw	TAGCACTAGAGAAAGAGGAGAAATACTAGATGGCCG AGGTGCAGCTGG	48bp	72	50	Uchino	Macrogen Japan Corp.
  scFv_ctl_Rv, CBDcex_ctl_Rv, CBDclos_ctl_Rv	CATCTAGTATTTCTCCTCTTTCTCTAGTGCTAGC	34bp	61	41	Uchino	Macrogen Japan Corp.
  CBDcex_ctl_Fw	TAGCACTAGAGAAAGAGGAGAAATACTAGATGGGTC CGGCCGGGTGCCA	49bp	74	53	Uchino	Macrogen Japan Corp.
  CBDclos_ctl_Fw	TAGCACTAGAGAAAGAGGAGAAATACTAGATGTCAT CAATGTCAGTTGAATTTTACAAC	59bp	67	34	Uchino	Macrogen Japan Corp.
  RFP_His_CBDcex.Rv	CCACAGCACCTGGCACCCGGCCGGACCATGATGATG ATGATGATGTTATTAAGCACCGGTGGAGTGACG	69bp	79	57	Uchino	Macrogen Japan Corp.
  RFP_His_CBDclos.Rv	GAGTTGTAAAATTCAACTGACATTGATGAATGATGA TGATGATGATGTTATTAAGCACCGGTGGAGTGACG	71bp	71	38	Uchino	Macrogen Japan Corp.
  INP primer for CBD.Fw	ACGACGACTGGATCGAAGTTAAAG	24bp	58	46	Miyazaki	Hokkaido System Science Co., Ltd.
  CBDcex primer.Rv	GCCGTTGAAGCCGAACTG	18bp	57	61	Miyazaki	Hokkaido System Science Co., Ltd.
  scFv primer1'Fw	TGCAACCTCTGCATTCATCTT	21bp	56	43	Miyazaki	Hokkaido System Science Co., Ltd.
  scFv primer2'Rv	CCCTGGCCCCAGTAGTCA	18bp	59	67	Miyazaki	Hokkaido System Science Co., Ltd.
  rRNA 16S forward primer1	GACGGGGGCCCGCACAAG	18bp	65	78	Miyazaki	Hokkaido System Science Co., Ltd.
  rRNA 16S reverse primer1	CTGGTCGTAAGGGCCATG	18bp	56	61	Miyazaki	Hokkaido System Science Co., Ltd.
  Prefix-F	GAATTCGCGGCCGCTTCTAG	20bp	60	60	iGEM	Hokkaido System Science Co., Ltd.
  Suffix-R	CTGCAGCGGCCGCTACTAGTA	21bp	62	62	iGEM	Hokkaido System Science Co., Ltd.
  INP_His_coding_Rv	ggaCTGCAGCGGCCGCTACTAGTACTAATGATGATG ATGATGATGggatccgcctcctgcaccg	64bp	78	56	Uchino	invitrogen
  INP_His_coding_Fw	ctgGAATTCGCGGCCGCTTCTAGAGatgaccctgga caaagctctgg	47bp	75bp	57	Uchino	invitrogen

  Materials

  Kit

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

  Restriction Enzyme

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

  Polymerase

  Name	Supplier
  KAPA™HiFi HotStart ReadyMix (2x)	KAPABIOSYSTEMS
  KAPA2G™ Fast HotStart ReadyMix with dye (2x)	KAPABIOSYSTEMS
  KAPATaq™EXtra HotStart ReadyMix with dye	KAPABIOSYSTEMS
  Quick Taq® HS DyeMix	TOYOBO

  DNA ligase

  Name	Supplier
  Ligation high Ver.2	TOYOBO

  Marker

  Name	Supplier
  1kb DNA Ladder	TaKaRa
  100bp DNA Ladder	TaKaRa
  1kb Plus DNA Ladder	Thermo Fisher Scientific

  Organism

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

  Antibiotics

  Name	Supplier
  Chloramphenicol	nacalai tesque
  Ampicillin Sodium Salt	nacalai tesque

  Equipment

  Name	Supplier
  Bemliese SR601	AsahiKASEI
  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
  Chemi Doc XRS+	BIO-RAD
  Cuvette	Eppendorf

  Backbones

  Name	Supplier
  pSB1C3	iGEM registry
  pSB1A2	iGEM registry
  pSB3C5	iGEM registry
  BBa_ J61002	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
  Tween-20	SANTA CRUZ
  Dimethyl sulfoxide	Wako

  SDSPAGE/Western blotting

  Name	Supplier
  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
  GII-4 rabbit anti-serum	Dr. Sano
  Amersham ECL Prime Blocking Reagent	GE healthcare
  Amersham ECL Prime WB Detection Reagent	GE healthcare
  Amersham ECL DualVue Western blotting Markers	GE healthcare
  WESTERN-VIEW™ Western Protein Size Marker(32-165kDa)	Wako

  Fluorescence Microscope

  Name	Supplier
  Immersion oil Type F	OLYMPUS JAPAN
  Filter Paper Qualitative 2 90mm	ADVANTEC
  Cellulose powder through 38μm(48mesh)	Wako
  5ml Polystyrene round-bottom tube with cell-strainer Cap	FALCON
  -Cellstain- DAPI solution	Doujindou

  Electronic Microscope

  Name	Supplier
  Paraformaldehyde(PFA)	Dr. Tadokoro
  Osmic acid	Dr. Tadokoro
  Acetone	Dr. Tadokoro

  Others

  Name	Supplier
  virus like protein GⅡ-４(Japan 2006)	Dr. Sano
  Norovirus like protein GⅡ-４(Narita 1997)	National institute of infectious diseases
  plasmid encoding human anti Norovirus scFv antibody 12A2 (pcpIII/12A2)	Dr. Moriguchi

  Methods

  Miniprep

  Minipreps were performed using FastGene™Plasmid Mini Kit according to the manufacturer's protocols.

  Gel Extraction

  Gel Extraction was performed using FastGene™Plasmid Mini Kit(GP3→MilliQ), and Wizard® SV Gel and PCR according to the manufacturer's protocols.

  Restriction Enzyme Digestion

  Restriction enzyme treatment was performed using Takara Bio's or Promega'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 protocols.

  Transformation

  1. Thaw competent cells on ice
  2. Add DNA sample (1~20μl) to competent cells. leave them on ice for 30 minutes
  3. Keep them in heating block for 45 seconds, then cool on ice for 2 minutes
  4. Add 40~200μl SOC liquid culture medium, then incubate with shaking for 1hour at 37°C
  5. Seed cells onto LB+antibiotics agar plate. Incubate for 24 hours at 37°C

  PCR

  PCR was performed using Wizard® SV Gel and PCR, KAPA™HiFi HotStart ReadyMix (2x) 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

  RT-PCR was performed using QuantiTect® Reverse Transcription Kit according to the manufacturer's protocol.

  Western blotting (basic protocol)

  1. Apply sample to SDS-PAGE minigel (BIOCRAFT 10%)
  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

  Western blotting (anti-NoVLP)

  Sample Preparation

  1. Mix MilliQ 12.5ul, SDS sample buffer 4.5 ul, and VLP 1 ul
  2. Vortex well and centrifuge with a minicentrifuge
  3. Follow the protocol for our basic Western Blotting

  Western blotting (Membrane fraction)

  Sample Preparation

  1. Cultivate 100 ml of E. coli culture whose OD600 values are 0.5
  2. Divide the 100 ml cell culture in two 50 ml tubes, centrifuge 5000 rpm for 10 min
  3. Wash with PBS 30 ml, centrifuge 5000 rpm for 10 min
  4. Resuspend with 50 mM Tris (pH8.0) 100 mM NaCl 10% glycerol 10ml
  5. Combine the same sample tubes and sonicate 10 min
  6. Centrifuge 4000 rpm for 10 min at room temperature
  7. Ultracentrifuge the supernatant at 45000 rpm (around 190000g) at room temperature, then remove the resulting supernatant
  8. Resuspend pellet in guanidine buffer (50 mM Tris (8.0) 300 mM NaCl 10 mM Imidazole 6M Guanidine-HCl 0.1% NP40 1mM DTT) to denature proteins
  9. Use Ni-NTA beads to purify the target protein
  10. Resuspend the purified protein in SDS-buffer
  11. Follow the protocol for Western Blotting

  Western blotting (Whole cell)

  Sample Preparation

  1. Cultivate 100 ml of E. coli culture overnight
  2. Pour 400 ul of the E.coli cell culture into 1.5 tubes
  3. Centrifuge 5000 rpm for 1 min and remove the supernatant
  4. Wash pellet with 1 ml of PBS
  5. Centrifuge 5000rpm for 1 min and remove the supernatant
  6. Add MilliQ 15 ul and SDS sample buffer 5 ul
  7. Vortex well and centrifuge with a minicentrifuge
  8. Sonicate for 10min
  9. Centrifuge with a minicentrifuge
  10. Follow the protocol for Western Blotting

  Growth Curve Drawing

  1. Measure OD600 of LB liquid medium (10μg/ml CP) and set blank
  2. Prepare 10ml LB liquid medium (10μg/ml CP) with E.coli sample whose OD600 values are adjusted to 1.0, Cover the tube with a plastic sheet with airholes
  3. Start incubating at 160rpm, 37°C. Measure the absorbance promptly and set it 0 minute
  4. Measure OD600 every 30 minutes (before 2hours)/every hour (after 2hours)

  Cellulose Affinity Assay with spectrophotometer

  1. Prepare LB +CP medium that contains objective E.coli, incubate overnight at 37°C
  2. Centrifuge this culture at 200g for 10 min.
  3. Collect the pellet, and add Carbonate bicarbonate buffer so that OD600 values come to about 0.2
  4. Take 1ml out of the cell suspension, and measure OD600 values. These OD600 values are <before>
  5. Incubate the cell suspension with 3cm by 2.5 cm cellulose sheet (BEMLIESE #606) for 1 h
  6. Measure OD600 values of the cell suspension. These OD600 values are <after>
  7. Transfer the cellulose sheet to a new buffer of the same volume, incubate for 1 hour
  8. Measure OD600 values of the buffer. These OD600 values are <wash>

  Cellulose Affinity Assay with Fluorescence Microscope

  Sample Preparation

  1. Centrifuge overnight cell culture at 200g for 20min
  2. Resuspend in PBS to OD600 of 0.2/1.0
  3. Mix 1ml cell suspension and cellulose powder/Bemliese
  4. Stir at room temperature, 800rpm for 6h
  5. Pellet with minicentrifuge
  6. Fix samples with 4% PFA for 10min
  7. Pellet with minicentrifuge
  8. Resuspend in PBS 500μl
  9. Repeat step 7 and 8 two more times
  10. Add 500μl 1ug/ml DAPI and leave for 15min
  11. 1Pellet with minicentrifuge
  12. 1Resuspend in PBS 500μl
  13. Repeat step 11 and12 two more times
  14. Filter sample with filtering membrane with 200g centrifugation for 5min
  15. Add PBS 500μl then centrifuge 200g for additional 5min
  16. Resuspend the remaining cellulose on filtering membrane in PBS 400μl, transfer to 1.5ml tube
  17. Pellet with minicentrifuge
  18. Resuspend in 20μl fluorescence fading inhibitor

  Statistics Analysis

  1. Randomly photograph at 600x the samples obtained from cellulose binding assay with a fluorescent microscope
  2. Measeure the total area of the cellulose using ImageJ, and count the number of E. coli bound to the cellulose
  3. Divide the number of E. coli binding to cellulose by the area (μm^2) of cellulose
  4. Conduct an F test on the cell number/cellulose(um^2) using Excel2016 MSO (16.0.6701.1041), and compare INPNC-His-ctl expressing E. coli with INPNC-His-CBDcex expressing E. coli
  5. Conduct Welch’s t-test (two-sided) and compare INPNC-His-ctl expressing E. coli with INPNC-His-CBDcex expressing E. coli

  Scanning Electron Microscopy (2015 Summer Experiment)

  Samples	Centrifugal Force
  INPNC-His-scFv(pSB3C5) 25μl+VLP2.5μl	200g
  BclA-His-scFv(pSB3C5)25μl+VLP2.5μl	200g

  Sample Preparation (E.coli+VLP)

  1. Prepare 45ml of sample E.coli liquid culture whose OD600 values are around 1.0
  2. Take 25μl, and pour into sample tubes
  3. Centrifuge VLP with minicentrifuge. After tapping it, pour 2.5μl into tubes
  4. Incubate for 24 hours
  5. Centrifuge at 200g for 10 minutes
  6. Suspend the pellets in 1ml PBS
  7. Centrifuge at 200g for 10 minutes
  8. Remove half of the supernatant, then resuspend with PBS

  Scanning Electron Microscopy (2016 Summer Experiment)

  Samples	Centrifugal Force
  INPNC-His-scFv(pSB3C5) 25μl+VLP2.5μl	3000g
  BclA-His-scFv(pSB3C5)25μl+VLP2.5μl	3000g
  INPNC-His-ctl 25μl+VLP2.5μl	3000g
  BclA-His-ctl 25μl+VLP2.5μl	3000g
  INPNC-His-scFv(pSB3C5) 25μl+VLP2.5μl	200g
  BclA-His-scFv(pSB3C5)25μl+VLP2.5μl	200g
  INPNC-His-ctl 25μl+VLP2.5μl	200g
  BclA-His-ctl 25μl+VLP2.5μl	200g

  Sample Preparation (E.coli+VLP)

  1. Prepare 45ml of sample E.coli liquid culture whose OD600 values are adjusted to 1.0
  2. Take 25μl aliquot, and transfer into sample tubes
  3. Centrifuge VLP with minicentrifuge. After tapping it, add 2.5μl into tubes
  4. Incubate for 24 hours
  5. Centrifuge at 200g/3000g for 10 minutes
  6. Suspend the pellets in 1ml PBS
  7. Centrifuge at 200g/3000g for 10 minutes
  8. Remove half of the supernatant, then resuspend in PBS

  Sample Preparation (PBS+VLP)

  1. After tapping VLP, pour 2μl into 200ml PBS
  2. Mark where pellets should have aggregated if E. coli were in the tube
  3. Centrifuge at 200g for 10 minutes
  4. Take 100μl of the supernatant, then pour into 1. tube

  Shape Analysis

  1. Determine aspect ratios of all E.coli from the SEM pictures using ImageJ
  2. Conduct an F test on the aspect ratios using Excel2016 MSO (16.0.6701.1041), and compare BclA-His-scFv expressing E. coli with INPNC-His-scFv expressing E. coli
  3. Conduct Welch’s t-test　 (two-sided) and compare BclA-His-scFv expressing E. coli with INPNC-His-scFv expressing E. coli