Biomod/2014/Kashiwa/Protocols

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<body> PROTOCOLS


<a href="#" onclick="HideCBox('CBoxBody1'); return false;" title="折りたたみ／復元">[show/hide]</a>

  Contents：

  • <a href=#1> 1. The Sensing System: The Receptor </a>
    • <a href="#1-1">1-1. Preparing the components </a>
      • <a href="#1-1-1">1-1(a). Folding the Wall </a>
      • <a href="#1-1-2">1-1(b). Producing MISTIC </a>
      • <a href="#1-1-3">1-1(c). Designing the Activator </a>
    • <a href="#1-2">1-2. Embedding the Wall into the liposome </a>
    • <a href="#1-3">1-3. Linking the Activator to the liposome </a>
    • <a href="#1-4">1-4. Combining the Wall and the Activator </a>
    • <a href="#1-5">1-5. Separating the Wall from the Activator </a>
  • <a href="#2">2. The Moving System: The Motor </a>
    • <a href="#2-1">2-1. Producing the Motor-Monomer </a>
      • <a href="#2-1-1"> 2-1(a). Folding the Motor-Monomer body </a>
      • <a href="#2-1-2"> 2-1(b). Synthesizing divalent SA </a>
      • <a href="#2-1-3"> 2-1(c). Equipping the Motor-Monomer with divalent SA </a>
    • <a href="#2-2">2-2. Deactivating and reactivating the binding capacity of streptavidin </a>
    • <a href="#2-3">2-3. Putting the Motor-Monomers into the liposome </a>




<a name="1-1"> </a>
<a name="1-1-1"> </a>

<a href=""> 1. The Sensing System: The Receptor</a>

1-1. Preparing the components

1-1(a). Folding the Wall

<tbody> </tbody>
Reagents (f. 10µL)
Staple mix5 µL
M13mp18ss4 µL
10 x monomer buffer*11 µL

*1 10 x monomer buffer (100 mL)

1.0 M Tris-HCl (pH 8.0) 10mL

1.0 M MgCl2 (f. 150 mM) 15mL

1.0 M NaCl (f. 25 mM) 2.5mL


1) Mix the reagents.

2) Anneal as follows

85°C, 5 min till 49.3°C, 2h till 4°C, forever

Analyze by 1 % agarose gel electrophoresis.

Stain the gel by EtBr for 30 minutes.

Take photo of the gel by LAS-4000.


1-1(b). Producing MISTIC

  • Protocol 1

We used MISTIC gene inserted in plDTSmart(kan) (IDT Co.). The sequences of primers are shown in note 2. Quick Change produce were transformed into XL10-Gold, andplasmid was prepared by QIAprep Spin Miniprep Kit (QIAGEN Co.).

Note 1: The base sequence of MISTIC (wild type)
ATGTTTTGTACATTTTTTGAAAAACATCACCGGAAGTGGGACATACTGTTAGAAAAAAGCACGGGTGTGATGGAAGCTATGAAAGT
GACGAGTGAGGAAAAGGAACAGCTGAGCACAGCAATCGACCGAATGAATGAAGGACTGGACGCGTTTATCCAGCTGTATAATGA
ATCGGAAATTGATGAACCGCTTATTCAGCTTGATGATGATACAGCCGAGTTAATGAAGCAGGCCCGAGATATGTACGGCCAGGAAA
AGCTAAATGAGAAATTAAATACAATTATTAAACAGATTTTATCCATCTCAGTATCTGAAGAAGGAGAAAAAGAATGA
Note 2: The sequence of primer
Forward:
GGGTGTGATGGAAGCTATGAAATGTACGAGTGAGGAAAAGGAACAG
Reverse:
CTGTTCCTTTTCCTCACTCGTACATTTCATAGCTTCCATCACACCC

  • Protocol 2

We referred to a thesis which we mentioned at reference No.2. The primer which we used is written on note 3. PCR was first conducted using primer3 (unnatural amino acids were introduced by using primer 3-1~3-7) and primer4, then primer1 and primer4 and finally in order to increase the concentration, primer2 and primer4. Electrophoresis was done by 1% agarose gel, 100V and 1 hour.

Note 3:
1.PURE_universal:
GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCA
2.PURE_amplify:
GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTC
3-0.Mistic_for_PURE:
TTTGTTTAACTTTAAGAAGGAGATATACCAATGTTTGTGACATTTTTTGAAAAACATCACC
4.Mistic_rev:
ATACATGAATGGATCCTTAATGGTGATGGTGATGGTGTTCTTTTTCTCCTTCTTCAGATACTG
3-1.Mistic_amber2_for_PURE:
CTTTAAGAAGGAGATATACCAATGTAGGTGACATTTTTTGAAAAACATCAC
3-2.Mistic_amber3_for_PURE:
CTTTAAGAAGGAGATATACCAATGTTTTAGACATTTTTTGAAAAACATCAC
3-3.Mistic_amber4_for_PURE:
CTTTAAGAAGGAGATATACCAATGTTTGTGTAGTTTTTTGAAAAACATCAC
3-4.Mistic_amber5_for_PURE:
CTTTAAGAAGGAGATATACCAATGTTTGTGACATAGTTTGAAAAACATCAC
3-5.Mistic_amber6_for_PURE:
CTTTAAGAAGGAGATATACCAATGTTTGTGACATTTTAGGAAAAACATCACCGG
3-6.Mistic_amber7_for_PURE:
CTTTAAGAAGGAGATATACCAATGTTTGTGACATTTTTTTAGAAACATCACCGGAAG
3-7.Mistic_amber8_for_PURE:
CTTTAAGAAGGAGATATACCAATGTTTGTGACATTTTTTGAATAGCATCACCGGAAGTGG

  • Protocol 3-1

We used PUREfrex® and Biotin-XX-AF (CloverDirect™) for unnatural amino acids. After electrophoresis, we rinsed and dried up the gel, copied it to IP for overnight, and scanned it to read the value of PSL by BAS.

  • Protocol 3-2

We used His Spin Trap (GE Co.) for purification. 18% acrylamide gel was used for SDS-PAGE and performed at 200V for 75 minutes. We stained the gel by SYPRO ORANGE. The composition of buffers is as follows.
・Binding[Wash] Buffer: 50mM Tris-pH8.0, 300mM KCl, 10mM imidazole, 51mM n-octyl-ß-D-glucopyranoside
・Elusion Buffer: 50mM Tris-pH8.0, 300mM KCl, 500mM imidazole, 51mM n-octyl-ß-D-glucopyranoside

  • Protocol 4

The procedure is the same as the one of the confirmation of the MISTIC expression. Electrophoresis was performed by 18% acrylamide gel, 200V and 75 minutes.

  • Protocol 5

The procedure is the same as the confirmation of the expression of MISTIC. Streptavidin beads (TAMAGAWA SEIKI Co.) were used for SA-bead and 2µL of 20mg/mL solution of them were added to the 10µL of PURE reaction solution. Electrophoresis was performed by 18% acrylamide gel, 300V and 40 minutes.

  • Protocol 6

We used ProbeQuant™ G-50 Micro Columns (GE Co.) for purification.

1-1(c). Designing the Activator

(i) Evaluation of Lambda Exonuclease activity

Procedure

1) Two ssDNA mixtures (a and a’ mixture ,and b and b’ mixture) were annealed at 65℃ for 5 minutes.

2) The two dsDNA reagents were mixed.

3) The dsDNA mixture and Lambda Exonuclease were mixed and incubated at 37°C.

4) The mixture was analyzed by 20% Native-PAGE.

5) The gel was stained by SYBR Gold for 15 minutes.

6) The gel was taken a photo by LAS-4000.

<tbody> </tbody>
Reaction Reagent
dsDNA mixture 4µL
Lambda Exonuclease5µL
10×reaction buffer1µL

10×reaction buffers in each experiment are as below.

1) 670mM Glycine-KOH(pH7.5 8.0, 8.5, 9.0, 9.4), 2.5mM MgCl2, 50&mibro;g/ml BSA

2) 670mM Glycine-KOH(pH8.0), 2.5, 5.0, 7.5, 10.0, 12.5mM MgCl2, 50µg/ml BSA

(ii) Oligo-modification of Lambda-Exonuclease

<tbody> </tbody>
Reagent
0.2M KPO4(pH7.8)5µL
200µM Cy3-modified oligo10µL
30mM BS(PEG)9 2µL

Procedure

1) The solution was mixed.

2) The mixture was incubated at room temperature for 5 minutes.

3) The solution was refines by NAP-5 column.

4) Lambda Exonuclease and refined oligo that are equimolecular amount were mixed.

5) The mixture was incubated on ice for 3 hours.

6) The solution was analyzed by 20% Native-PAGE and 20% SDS-PAGE.

7) The Native-PAGE gel was taken a photo by LAS-4000.

8) The SDS-PAGE gel was stained by SYPRO Orange for 60 minutes and taken a photo by LAS-400.

(iii)Oligo-modification of HindⅢ

Exchange-buffer

<tbody> </tbody>
Reagents (f: 50ml)
0.2M HEPES (pH8.5) 5ml
5M NaCl 500ul
1M DTT 50ul
0.5M EDTA 10ul
MQ up to 50ml

Reagents for Oligo-BS(PEG)9

0.2M KPO4 5ul

200uM cy3-Oligonucleotide 10ul

20mM BS(PEG)9

Reagents for Oligo- BS(PEG)9-HindⅢ

Fraction 20ul

HindⅢ 20ul



1-2. Embedding the Wall into the liposome

(i) Hybridization of cholesterol oligonucleotide with Wall

In this experiment, we optimized the concentration of the cholesterol oligonucleotide and the number of handles, which is necessary for wall penetration into liposome. The result was assayed by 1% agarose gel electrophoresis.

<tbody>

</tbody>

Reagents
Purified Wall (0.03µM)6µL
Cholesterol oligomer 1.5µL

Sample2 and 3 do not have handles for the cholesterol oligonucleotide to hybridize. Sample 5 and 6 have 4, sample 8 and 9 have 10, sample 11 and 12 have handles for the cholesterol oligonucleotide to hybridize.

<tbody>

</tbody>

Concentration of cholesterol oligonucleotide
Sample2_1, 3_1, 5_1, 6_10.24µM
Sample2_2, 3_2, 5_2, 6_20.48µM
Sample2_3, 3_3, 5_3, 6_32.4µM
Sample2_4, 3_4, 5_4, 6_44.8µM
Sample8_1, 9_10.60µM
Sample8_2, 9_21.2µM
Sample8_3, 9_36.0µM
Sample8_4, 9_412µM
Sample11_1, 12_11.14µM
Sample11_2, 12_22.28µM
Sample11_3, 12_311.4µM
Sample11_4, 12_422.8µM

Procedures

Reagents are mixed and incubated for 60 minutes at room temperature.

(ii) Putting the Wall into the liposome

Same as <a href="#2-3">2-3. Putting the Monomer into the liposome</a>.


1-3. Linking the Activator to the liposome

1-4. Combining the Wall and the Activator

1-5. Separating the Wall from the Activator

<a href=""> 2. The Moving System: The Motor</a>

2-1. Producing the Motor-Monomer

2-1(a). Folding the Motor-Monomer body

<tbody> </tbody>
Reagents (f. 10µL)
Staple mix5 µL
M13mp18ss4 µL
10 x monomer buffer*11 µL

*1 10 x monomer buffer (100 mL)

1.0 M Tris-HCl (pH 8.0) 10mL

1.0 M MgCl2 (f. 150 mM) 15mL

1.0 M NaCl (f. 25 mM) 2.5mL

1) Mix the reagents.

2) Anneal as follows

85°C, 5 min till 49.3°C, 2h till 4°C, forever

Analyze by 1 % agarose gel electrophoresis.

Stain the gel by EtBr for 30 minutes.

Take photo of the gel by LAS-4000.

2-1(b). Synthesizing divalent SA

2-1(c). Equipping the Motor-Monomer with divalent SA

(i) Modifying divalent SA with NHS

Procedure

1) Exchange the buffer of divalent SA to the Exchange-buffer by NAP-5.

2) Mix divalent SA with Cy5-NHS in tetraborate and incubate for 1 hour at room temperature.

3) Add biotin-oligonucleotide to the mixture and incubate 30 minutes at room temperature.

4) Analyze by Native-PAGE (10% Resolving Gel)

<tbody>
Exchange-buffer
0.2M HEPES (pH8.5) 5ml
5M NaCl 500µl
1M DTT 50µl
0.5M EDTA 10µl
MQ up to 50ml
<tbody> 　 </tbody>
Reagents for SA and Cy5-NHS
0.45µM divalent SA 10µL
Cy5-NHS10µL
tetraborate (pH 8.1) 5µL


Reagents for Cy5-SA-biotin-oligonucleotide:

Fraction 5µl

the threefold amounts of biotin-oligonucleotide 5µl

(ii) Click reaction

Procedure

1) Mix click reagent and NH2-modified oligonucleotide in tetraborate (pH8.1) and incubate for 1h at room temperature.

2) Refine with QIA quick Nucleotide Column.

3) Add 450µL of PNI buffer to the mixture.

4) Apply to column.

5)Centrifuge at 6krpm for 1minutes.

6) Add 750 µL of buffer PE.

7) Centrifuge at 6krpm for 1minutes.

8) Add 750 µL of buffer PE.

9) Centrifuge at 6krpm for 1minutes.

10) Centrifuge at 13krpm for 1minutes.

11) Put the column on new tube and add 33µL of MQ and incubate for 3 minutes.

12) Centrifuge at 13krpm for 1minutes.

13) Dilute the mixture with MQ to the appropriate concentrate.

14) Mix one of Azide group and that of Alkyne group and incubate at room temperature.

15) Analyze by 15% Urea gel electrophoresis.

<tbody> </tbody>
Reagent of click reagent and NH2-modified oligonucleotide
10mM click reagent 7µL
tetraborate(pH8.1) 3µL
2µg/µL　NH2-modified oligonucleotide 2µL
<tbody>

2-2. Deactivating and reactivating the binding activity of streptavidin

(i) Make Biotin-Beads

Reagent of click reaction
6µM Azi-oligo 5µL
6µM Alk-oligo 5µL
<tbody>

</tbody>

Reagents (f: 20uL)
HEPES-KOH (pH8.5, f.90 mM) 9.0 µL
Biotin and amino modified Oligonucleotide (f: 65 µM) 1 µL
NHS-Beads (f.10 mg/ml) 10 µL

Procedure

Mix NHS-Beads with the oligonucleotide which has Biotin and amino group Remove the solvent.

(ii) Deactivate and reactivate SA

<tbody> </tbody>
Reagents for SA Deactivation (f: 10µL)
Ds DNA of Leader and Blocker (f: 5µM) 2.0µL
Divalent SA (f: 30µM) 8.0µL
Biotin-Beads 0.2mg
1Ds DNA (f: 10µL)
Biotin modified Oligonucleotide (Leader) (f: 25µM) 5.0µL
Desthiobiotin modified Cy3-Oligonucleotide (Blocker) (f: 25µM) 5.0µL
<tbody> </tbody>
Reagents for cutting dsDNA on SA (f: 15µL)
Purified Deactivation SA-dsDNA complex 4µL
HindⅢ (f: 6700U/ml) 1uL
10×NEB Buffer 2.1 1.5µL
MQ 8.5µL
<tbody> </tbody>
Reagents for cutting ds DNA on SA (f: 10uL)
Purified Deactivation SA-dsDNA complex 2 µL
Biotin modified Cy5-Oligonucleotide (Chaser) (f: 5 µM) 2.0 µL
MQ 6 µL
<tbody>

</tbody>

Reagents for reactivation of SA (f: 10 µL)
dsDNA cutted SA-dsDNA complex（solution） 8µL
Biotin modified Cy5-Oligonucleotide (Chaser) (f: 5 µM) 2.0µL

Reagents for Native-PAGE

15 % Resolving Gel

MQ 4.8 ml

30% Acrylamide mix 10 ml

1.5M Tris-HCl (pH 8.8) 5.0 ml

10 % APS 0.2 ml

TEMED 8.0 ul

5 % Stacking Gel

MQ 2.79 ml

30% Acrylamide mix 0.67 ml

1.0 M Tris-HCl (pH 6.8) 0.5 ml

10 % APS 40 ul

TEMED 4.0 ul

Procedure

(i) Make Biotin-Beads

(ii) Deactivate and reactivate SA

1) Anneal dsDNA of Leader and Blocker (95°C, 3min to 4&degC, forever)

2) Mix the dsDNA with divalent SA at 4℃ for 30min

3) Remove SA which is not bound to the dsDNA by Biotin-Beads

4) Cut the dsDNA bound to SA by HindⅢ

5) Add Chaser to the SA of which the dsDNA was cut

Analyze

1) Analyze by Native PAGE (15% Resolving Gel)

2) Observe with LAS-4000 and take pictures


2-3. Putting the Motor-Monomers into the liposome

(i) Binding Motor-Monomers and Qdots/p>

Reagents

Motor-Monomers (unpurified) 80µL

Qdot 655 streptavidin (1µM) 6.4µL

Procedure

1) 80µL of Motor-Monomers and 6.4µL of Q-dot were mixed and incubated at room temperature for 30 minutes.

2) The mixture was purified.

(ii) Preparation of GUVs

Reagents

Lipid mix (3mM)

POPC 12mg

Paraffin 5mL

Outer solution

Glucose (400mM) 250µL

Tris (50mM, pH 7.5) 250µL

Emulsion mix

Lipid mix (0.5mM) 500µL

Inner solution(200mM) 10µL

Inner solution

1. GUV including DNA constructs

Mercaptoethanol (4mM) 5.0µL

DNA constructs with Qdot 2.5µL

Sucrose (1M) 2.0µL

Tris (500mM, pH 7.5) 0.5µL

2. GUV not including Motor-Monomers or Walls

Sucrose (400mM) 5µL

Tris (50mM, pH 7.5) 5µ

Procedure

1) Take Glucose (500µL) into a tube as outer solution.

2) Add Lipid mix (0.5mM) on the glucose solution carefully.

3) Add Emulsion mix on the top and rapidly centrifuge at 4°C, 1800rpm for 10 minutes.</o>

4) Centrifuge again at 4°C, 5000rpm for 10 minutes.

5) The upper layer is removed and GUVs are collected using pipetman.

6) 5µL of GUV was mixed with 0.5µL of Nile Red (10µM) and observed by confocal microscope.




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