Biomod/2011/TeamJapan/Sendai/Notes

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<ul id="verticalmenu" class="glossymenu"> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai">Home</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Strategy">Strategy</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Design">Design</a></li> <li><a href="#">Experiments</a> 

   <ul>
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Electrophoresis">Electrophoresis</a> 
   <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Results/Atomic_Force_Microscope">AFM</a> 
   </ul>

</li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Computational_design/Simulation" >Simulation</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Notes">Notes</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Team">Team</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Resources">Resources</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Sendai/Sitemap">Sitemap</a></li>


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Brainstorming

Figure 1.Brainstorming

To improve the efficiency of the molecular robot we gathered many ideas related with the robot design and its mechanism for movement:

Hexagonal prism

By design the motion of this kind of robot is rolling rather than walking. This robbot was our first plan.

Advantage

  • A maximum of 72 legs can be attached to a hexagonal prismatic body.
  • The robot does not go backwards. Therefore, walking forward efficiently.

Disadvantage

  • The difficulty in verifying the 3D body design.
  • The time cost that is necessary to get the robot annealed.
  • The complexity of the structure due to the different variety of staples.

Here you can find this robot sequence and more information.


Wall runner

Over the path we can create two walls that link the start point and goal, analogous to a simple labyrinth.

Advantage

  • This robot will always go towards one direction.
  • A novel mechanism for motion.

Disadvantage

  • This robot may fall at the time of observation by AFM.
  • The difficulty to make a single wall.


Sticky motif robot

This design is a very simple one, where it is combined only four DNA double strands.
The sticky motif robot was our plan B in case we failed to produce the triangular prism robot.
In addition the legs design include both kind of robots for saving time and costs.
Here you can find this robot sequence and more information.


ぼくたちがいままでやったこと The progress of our activity

六角柱 The hexagonal prism

本体のみをアニーリング Annealing of robot’s body
電気泳動 Electrophoresis

三角柱 The triangular prism

本体のみをアニーリング Annealing of robot’s body
電気泳動 Electrophoresis
本体足つきをアニーリング Annealing of robot’s body and legs
余分なM13を切る Cutting of excessive M13

足 Legs

フィールドをアニーリング Annealing of field
スパイダーによるフィールドと足の切断実験 Cutting of leg and substrate with spider
フィールドに乗せる実験 Putting robot on field

実験方法 The method of experiments

アニーリング Annealing

Freeze'n squeeze

Our experiment refers to Harvard team wiki. Thank you Harvard team!!


足の切断実験 Cutting of leg and substrate

蛍光付きのsubstrate Substrate with fluorescence

（spiderの論文を参照した配列に蛍光と消光をつけたsubstrateとlegが二重螺旋を組み、Zn2+入りのbufferを入れることで蛍光と消光剤が離れ結果的に蛍光が光っていることにより足が切断されていることがわかる。以下にその手順を示す。）

Leg and substrate, which attached fluorescence and quencher, have constructed the double helix. By putting in buffer containing Zn2+, fluorescence is separated from quencher, and fluoresce. Therefore, by observing fluorescence, it can be confirmed whether legs and substrate were cut. The process is shown below.


（1．蛍光付きのsubstrateとlegを100nMに希釈してそれらを22μLずつ入れてbufferとして1×TA Mg2+と1×TA Mg2 with Zn2+(1mM,2mM,10mM)を入れてbufferを入れてからの時間を0min,5min,30minとし泳動用のbufferとして1×TA Mg2+を選んだ。電気泳動の結果からsubstrateのみとlegのみをsampleとして入れなかったためバンドが何を示しているかがわからず、また常温で正確に2本鎖を組んでいるかがわからないため次の泳動実験ではそのようなことを考慮した。）

１回目

gel:24% Poly-Acrylamide Gel

buffer:1×TAE Mg2+

sample:すべてlegとsubstrateが入っている bufferは①1×TAE Mg2+②⑤⑧1×TAE Mg2+with Zn2+1mM③⑥⑨1×TAE Mg2+with Zn2+2mM④⑦⑩1×TAE Mg2+with Zn2+10mM buffer入れてからの経過時間②③④0min⑤⑥⑦5min⑧⑨⑩30min


1.Leg and substrate with fluorescence were diluted to 100nM, and they were taken every 22 micro / L. As a buffer, 1xTA Mg2+ and 1xTA Mg2+ with Zn2+ (1mM, 2mM, 10mM) were put in. Time after putting in buffer was set to 0min, 5min, and 30min. As a buffer for electrophoresis, 1xTA Mg2+ was used. As a result of electrophoresis, it did not turn out what the bands would show, since only substrate and only leg were not put in as sample. Moreover, there is no telling whether leg and substrate have constructed double helix correctly at normal temperature. So such things were taken into consideration in the next electrophoresis.


（2．新しいsampleとしてsubstrateとlegを100nMに希釈してそれらを22μLずつ入れてから熱湯に2～3min漬けて冷水に10分ほど浸し確実に2本鎖を組ませてからbufferを入れるもの、またsampleの濃度が濃すぎたためsubstrateとlegが切れても近くにいるため 蛍光が反応しなかったかもしれないと考え濃度を1/3にしたものを新しいsampleとして加えた。電気泳動の結果からおそらくすべてのsampleにおいて切断されたと確認できるため、Zn2+入りでなくともMg2+入りのbufferが作用することでsubstrateとlegが切断されてしまうと考えられる。また熱処理を加えなくてもきちんと2本鎖を組み、濃度を薄くしなくても切断されていることが十分に確認できた。そのため原因としてはMg2+が作用していることが考えられるため、gelと泳動用のbufferでMg2+抜きのbufferを使用することでMg2+の効果を観察することを次の実験で考慮した。）

２回目

gel:24% Poly-Acrylamide Gel

buffer:1×TAE Mg2+

sample:①only leg ②only substrate ③⑤⑦⑨1×TAE Mg2+　④⑥⑧⑩⑪1×TAE Mg2+ with Zn2+1mM ③④buffer入れて0min ⑤⑥buffer入れて15min　⑦⑧熱処理をしてbuffer入れて15min　⑨⑩濃度を1/3にしてbuffer入れて15min　⑪濃度を1/3にして熱処理をしてbuffer入れて15min


2.As a new sample, sample-A and sample-B were added. Sample-A : The substrate and leg are diluted to 100nM and they are put in every 22micro / L . After dipping in boiling water for 2~3 minutes, it dips in cold water for 10 minutes. Buffer is put in after making 2 chains construct certainly. Sample-B : Concentration is set to one third. Because it thought that substrate and leg which were separated approached closely since concentration was too dense, and fluorescence can not react. As a result of electrophoresis, cutting can be checked in all the samples. Therefore, it is thought that substrate and leg will be cut by acting of Mg2+ even if Zn2+ is not included. Moreover, even if heat treatment did not added, the double helix was constructed exactly, and even if it does not make concentration thin, cutting has fully checked. It is considered as a cause that Mg2+ is acting. In the next experiment, the effect of Mg2+ was observed by using buffer which does not contain Mg2+ for gel and electrophoresis.


(3．resultに記述したがMg2+が入っているbufferを用いるとsubstrateとlegが切断されていることがわかる。Zn2+の作用としては0minでの輝度差からZn2+が入っていると切断の初速度が多少早くなることがわかった。)

3.By putting in buffer containing Mg2+, legs and substrate were cut (refer to Results). As an action of Zn2+, it turned out from the difference of luminosity in 0min that the initial velocity of cutting becomes quick a little by using buffer containing Zn2+.


蛍光無しのsubstrate　Substrate without fluorescence

(蛍光ありのsubstrateでは染色液を使わなかったので、蛍光無しのsubstrateで染色をした場合切断がわかるかどうかを確認したかった。蛍光ありでの電気泳動と条件を変えずに結果が得れたので良い。)

Since a stain solution was not used in the experiment using substrate with fluorescence, in order to investigate whether it can be checked that reg and substrate have been cut even when a stain solution is used, carried out experiment using substrate without fluorescence.


M13の切断　Cutting of M13

To cut m13, specific part of m13 sequence form a double helix, secondly react by enzyme, finally clean up of discarded enzyme and pick out only M13.The process is shown below.


Japanese (M13を切断するためには特定の部分の配列を二重螺旋にしてから酵素を反応させて切断し、その後不要な酵素などを除去してM13のみを取り出す。以下にその手順を示す。)

Form a double helix

Mix 5μL M13(84nM), 3μL DNA that specific part of m13 sequence form a double helix(5μM), 2μL Buffer BSA, 2μL BalⅠ buffer, and 5μL mQ. Set in PCR, the condition is 3min at 95°C, 3min at 65°C, for the duration of enzyme reaction, let the sample at 37°C.


Japanese (M13(84nM):5μL M13と特定の部分で相補鎖を組ませるようなDNA2種類(5μM):3μL BSA:2μL BalⅠ buffer:2μL mQ:5μL
これらを混ぜてサーマルサイクラーに入れる。その時の条件はを95°Cを3分、65°Cを3分、その後は酵素を働かせる間は37°Cを保つ。)


Cutting M13 by enzyme reaction

Add 1μL BalⅠin the sample, let the sample stand for 2 hours at 37°C.
Add 3μL 10×H buffer and 7μL mQ, add 1μL PstⅠ, let the sample stand for 1 hours at 37°C.


Clean up of enzyme

The first method
  • All centrifugation steps are carried out at 16,000×g(13,000 rpm) in a conventional tabletop micro-centrifuge at room temperature.
Add 5 volumes of Buffer PBI to 1 volume of the cutting M13 and mix.
Place a column in a 2ml collection tube.
To bind DNA, apply the sample to the column and centrifuge for 1min.
Discard flow-through.Place the column back into the same tube.
To wash, add 0.75ml Buffer PE to the column and centrifuge for 1min.
Discard flow-through and place the column back in the same tube. Centrifuge the column for additional 1 min.
Place column in a clean 1.5ml micro-centrifuge tube.
To elute DNA, add 25 μL Buffer EB (10mM Tris-Cl, pH8.5) to the center of the column, let the column stand for 1 min, and then centrifuge for 1 min.
The first method is to make sure of cut M13, so this method is mixing long cut M13 and short(we want) cut M13.


The second method
  • All centrifugation steps are carried out at 16,000×g(13,000 rpm) in a conventional tabletop micro-centrifuge at room temperature.
Add 1/10 volumes of Sodium Acetate, and 2.5 volumes of ethanol to 1 volume of the cutting M13 and mix, and then centrifuge for 10 min.
Discard supernatant being careful not to throw out DNA pellet.
Dissolve pellet in 1×TE buffer.
After electrophoresis, Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
Weight the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel.
Incubate at 50°C for 10 min. To help dissolve gel, mix by vortexing the tube every 3 min during the incubation.
After the gel slice has dissolve completely, add 1 gel volumes of isopropanol to the sample and mix.
Place a column in a provided 2ml collection tube.
To bind DNA, apply the sample to the column, and centrifuge for 1 min.
Discard flow-though and place column back in the same collection tube.
Add 0.5ml of Buffer QG to column and centrifuge for 1 min.
To wash, add 0.75 ml of Buffer PE to column and centrifuge for 1 min.
Discard the flow-though and centrifuge the column for an additional 1 min.
Place column into a clean 1.5 ml micro-centrifuge tube.
To elute DNA, add 25 μL Buffer EB (10mM Tris-Cl, pH8.5) to the center of the column, let the column stand for 1 min, and then centrifuge for 1 min.
The second method can purify only short(we want) M13.

PEG(polyethylene glycol)precipitation

We carried out PEG precipitation to get rid of the over staples from sample.
PEG precipitation

experimental condition

  • 10mM MgCl2,PEG6000 10%, to a sample.
  • At 16,000×g in a conventional tabletop microcentrifuge for 10 minute at RT.
  • Removed Supernatant liquid and a sample is dissolved by 1×TAE-Mg2+ of 80% of a total amount.


It seems that most staples are removed by two PEG precipitation.

二回PEG沈をすると、ステイプルはほとんど除去できるようである。

molecular spider's additional experiment スパイダー追試実験

For practice to observe by AFM, we carried out experiment about molecular spider. AFM観察の練習のために、spiderの作成と観察を試みた。

  • STV:C-Leg=1:4の作成法

STV 5mg/ml in K2HPO4:KH2PO4=5:11 pH6.5　　2.1μl
Capture-Leg 16nM 8μl
Mg2+ 1M 0.6μ
Tris:Tris-HCl=2.5:7.4 pH7.4 1μ
MQ 38.3μl
In this case,all is 50μl.


DNA sequence

The triangular prism sequence

Figure: triangular prism sequence of staples.
red for leg, green for prism, purple for capture-leg

ACTCACATTAATTGCGCCTGTCGTGCCAGCTGGGTGGTTTT
AGCCTGGGGTGAACGCGCGGGG
GCCGGAAGCATAAAGTGAAATTGTTATCCGCTTAGCTGTT
ATTACCGCCAGCCATTAAACGCTCATGGAAATGATTATTT
GTAGAAGAACTCAAACCGAGTAAAAGAGTCTGAGAATCTTG
TTAGTAATACCGTTGTAGCAATAC
CTTTCCAGTCGGGAAATTGCGCTCACTGCCCGTTCTTTGA
ATCGGCCCCTAATGAGTGAGCTA
CACAGGAAGCAACACAACATACGA
TTTGACGCTAATATCCAGAACAAT
AGTGAGGCCACTATCGGCCTTG
CAAATTAAACATCACTTGCCTGA
TCTTTTCCACCGCCTGGCCCTGATTGATGGT
AGAGGCGGTTTGGTCCACGCTG
TCCTGTGTGATGGCCCACTACGTGAACCGTCT
ACATTGGCAAGTTTTTTGGGGTCGAGGGAGCC
AGAAGTGAACGTGCTTTCCTCGTACAGGGCGC
GATTTTAGGCTAAACAGGAGGCCG
TGCCCTTACCAGCGAGACGGGCAATTAAAGG
AGCAAGCGCGTATTGGGCGCCAGCATTAATGA
CCCAAATCAGATTAATCATGGTCACACAATTC
TAAAGCACTCAATCGTCTGAAATGACCTACAT
CGAGCACGTATTTTTTATAATC
AGCGGGAACAGGAACGGTACGCCTCCATCACG
GGTTCCGAAGCCCGAGATAGGGTT
GTTTGCCCCAGTGGAACAAGAG
ATCAGGGCCTATTAAAGAACGTGG
CCCGATTTGGGGAAAGCCGGCGAA
GTACTATGGAGCGGGCGCTAGGG
ACCACCACACCCGCCGGGTCACGCTGCGCGTATATAAATC
AAAAGAATAATCGGCAAAATCCCTACAGCTGAT
GAGTGTTGTTCCAGTTCAGGCGAAAATCCTGTGAGAGTTGC
ACTCCAACGTCTTGACAGAGCAAAGGGCGAAAAACCATCA
CGTGGCGAGAAAGGAATAAATCGGAACCCTAAAGGTGCCG
AGAAAGCGAAAGGTTGCTTTGA
CGCTGGCAAGTGTAGCCGCTTAATGCGCCGCTTGGAATCAG

Field　staple's sequence

Substrate AGGACTTGGACACTAGGTACTTTTTCACTATAGGAAGAG


Figure. design of AFM Field
Figure. Field & substrate & leg

Stage1 AGGAAACCAGATAGCCGAACAAAG

Stage2 TGAGGGAGACAAAAGGGCGACATTTTTTTTACCAGA

Stage3 GAATAGAACTTTCAACAGTTTCAGTTTTCAACCGAT

Stage4 GGTTTATCCCAAAAGGAGCCTTTATTTTCGGAGTGA

Stage5 TCATGAGGAGGCTTTGAGGACTAATTTTATTGTATC

Stage6 CATCTTTGGGCAAAAGAATACACTTTTTAGACTTTT

Stage7 TAATCTTGAGGCTGGCTGACCTTCTTTTAAAACACT

Stage8 GAGTAGTATGCCCTGACGAGAAACTTTTATCAAGAG

Stage9 CCAAAAGGAATTACGAATGCAGATACATAACGTTTTACCAGAAC

Stage10 AAGTAAGCGAGGAAACGCAATAATTTTACCAG

Stage11 CGCCAAAGGGAAGGTAAATATTGAGGATTTTG

Stage12 CTAAACAAAGGAACAACTAAAGGACTCCAAAA

Stage13 AAAAGGCTAGCTTGCTTTCGAGGTGTAGCAAC

Stage14 GGCTACAGAAGTTTCCATTAAACGAACCTAAA

Stage15 ACGAAAGAACCCCCAGCGATTATATACAGACC

Stage16 AGGCGCATACAAGAACCGGATATTTTCAGTGA

Stage17 ATAAGGCTAATTGGGCTTGAGATGATACCACA

Stage18 TTCAACTAGGCATAGTAAGAGCAACACTATCA

Stage19 ACCCAAAATCTTACCGAAGCCCTTTTTAAGAA

Stage20 ATTCATTATAGAAAATTCATATGGAACGGAAT

Stage21 TAATAATTATGAATTTTCTGTATGCGGAAATT

Stage22 TAAACAGCTTTTCACGTTGAAAATATTGCGAA

Stage23 ACGTAATGCAGCATCGGAACGAGGGAATTTCT

Stage24 GAAACAAACCACTACGAAGGCACCGGTAAAAT

Stage25 AAATCAACGGACAGATGAACGGTGCCAAGCGC

Stage26 TCAACTTTGTAACAAAGCTGCTCACATTACCC

Stage27 TAACCCTCCAGTTGAGATTTAGGAGTTTAATT

Stage28 AATAGCTAGAACTGGCATGATTAAATTTTGTC

Stage29 ACAATCAAAAGGTGAATTATCACCTTCCAGAC

Stage30 GTTAGTAAATTTTGTTAAAATTCGAATTGTAA

Stage31 ACGTTAATTTGATACCGATAGTTGCCTCAGCA

Stage32 GCGAAAGACCTCATATATTTTAAATAAAAATT

Stage33 TTTAGAACGTACAACGGAGATTTGGACCAACT

Stage34 TTGAAAGATTCATTCCATATAACATACGGTGT

Stage35 CTGGAAGTAATCATTGTGAATTACAGGTAGAA

Stage36 AGATTCATGTTTACCAGACGACGATAAAAACC

Stage37 ATTACGCAAAGAGCAAGAAACAATGAAATAGC

Stage38 CTTGAGCCACCACGGAATAAGTTTGACTCCTT

Stage39 TTTTGTTATAAAGTTTTGTCGTCTGTCACCGA

Stage40 ATGACAACTATAAGCAAATATTTACATTAAAT

Stage41 CTGAGTAATTTGCGGGATCGTCACCGCCGACA

Stage42 CCTGATAATATTTCAACGCAAGGATGCAATGC

Stage43 CCAATTCTATAAGGGAACCGAACTTATCATCG

Stage44 ATTTTAAGAAATATGCAACTAAAGGTTGATTC

Stage45 AAAATAGCGGAACAACATTATTACCTTATGCG

Stage46 CCAATAATGTATGTTAGCAAACGTAAAGAAAC

Stage47 GCAAAGACATTTGGGAATTAGAGCAGTTAGCG

Stage48 TAACGATCAATCAGCTCATTTTTTAAAAACAG

Stage49 GTACCTAGTGTCCAAGTCCT GAAGATTGAACCATCGCCCACGCAGGAGTTAA

Stage50 AGGCCGCTTGTGTAGGTAAAGATTTTGCGGGA

Stage51　　 GAAGCCTTATTGTGTCGAAATCCGGCGCAGACTTTTTTTTTTTTTTTTTTTTGATGTCTACTTGCGTCAGGTTCTCGGC

Stage52 GGTCAATCGCGAACGAGTAGATTTTAGCTCAA

Stage53 CATGTTTTAACTGGCTCATTATACAATAAAAC

Stage54 GAACTAACTAAATCAAAAATCAGGTCTTTACC

Stage55 CATACATATAACCCACAAGAATTGAGTTAAGC

Stage56 TCACCAGTAAGGTGGCAACATATAAGAAAATA

Stage57 GGAACGCCCACAGACAGCCCTCATCAGCAAAA

Stage58 GTACCTAGTGTCCAAGTCCT ATATTCGGTAATCAGAAAAGCCCCAACCAATA

Stage59 TGAGAAAGTCGCTGAGGCTTGCAGTAACCGAT

Stage60 TCCATGTTTGACCCTGTAATACTTCAAAAGGG

Stage61 CATTAGATACTTAGCCGGAACGAGCGACCTGC

Stage62 ACGTTGGGGCTGAATATAATGCTGAGTTTGAC

Stage63 CTGACTATAAGAAAAATCTACGTTCAGTCAGG

Stage64 CAGAGAGACTTAGGTTGGGTTATACCTTTTTA

Stage65 ACCTCCGGAGCACCATTACCATTAAACGCCTG

Stage66 TAGCATTCATCAAAAATAATTCGCTATGTACC

Stage67 CCGGTTGAGGCTATCAGGTCATTGTTGAGAGA

Stage68 TCTACAAAGCCGGAGACAGTCAAATGTACCAA

Stage69 AAACATTAACATCCAATAAATCATAATAGTAG

Stage70 TAGCATTAACATTTCGCAAATGGTGGCTTAGA

Stage71 GCTTAATTGACCGGAAGCAAACTCGCTTCAAA

Stage72 GCGAACCATATAGTCAGAAGCAAAGCGGATTG

Stage73 TGTAAATGCAGAGGGTAATTGAGCGCTAATAT

Stage74 GGAAACGTTAGGTCTGAGAGACTATAACTATA

Stage75 TTCCTGTACACCAGTACAAACTACGCAAGGCC

Stage76 TCTGGAGCACTAGCATGTCAATCAGTCTGGCC

Stage77 AATATGATGAGAGGGTAGCTATTTCCTGAGAG

Stage78 GGCAAAGAATAAAGCTAAATCGGTTCACCATC

Stage79 TGTTTAGCTGGCATCAATTCTACTACAGGCAA

Stage80 CAGGATTAGGTCATTTTTGCGGATCAATAACC

Stage81 CATCAAAATCGCGTTTTAATTCGACAACAGGT

Stage82 AACAAAGTCTGATGCAAATCCAATGAATTTAT

Stage83 GTACCTAGTGTCCAAGTCCT CAAAATCACACCAATGAAACCATCTAACACTG

Stage84 GTACCTAGTGTCCAAGTCCT AGTTTCGTGCCAGCTTTCATCAACGAACGGTA

Stage85 ATCGTAAAAAACAAGAGAATCGATTGATAAAT

Stage86 TAATGCCGATTCAACCGTTCTAGCAATAAAGC

Stage87 CTCAGAGCATTAGCAAAATTAAGCGCGCGAGC

Stage88 TGAAAAGGTATATTTTCATTTGGGGCTCCTTT

Stage89 TGATAAGAGAGAGTACCTTTAATTCGAAAGAC

Stage90 TTCAAATAAGATTAAGAGGAAGCC

Stage91 AAAGAACGCGGGAGAATTAACTGAACACCCTG

Stage92 CACCGTAAGAGAAGAGTCAATAGTCGCAAGAC

Stage93 ATTAAATGTGAGCGAGAGGAACCCATGTACCGGATAGCAG

Stage94 GCATTAGACGAGAAAACTTTTTCACTTAGATT

Stage95 AAGACGCTTCAGTAGCGACAGAATGGATAGCA

Stage96 AGCCCAATTAACAACCCGTCGGATTCTCCGTG

Stage97 TTAGTTAAAGAATAACATAAAAACAGGGAAGC

Stage98 CCTTTAGCGAAAACATAGCGATAGAATATATT

Stage99 GGAACAAACCACCCTCATTTTCAGCAAGTTTG

Stage10 TTACAGAGTTTCATCTTCTGACCTTTCCCTTA

Stage101 GAATCCTTGTCAGACTGTAGCGCGGCCACCCT

Stage102 CAGAGCCACGGCGGATTGACCGTAATGGGATA

Stage103 GTACCTAGTGTCCAAGTCCT TGGTTTGACGTCAAAAATGAAAATAGCAGCCT

Stage104 GTACCTAGTGTCCAAGTCCT GGCATTTTTCGCTATTAATTAATTAAATTTAA

Stage105 GGTCACGTCGCCACCCTCAGAACCTTTTCATC

Stage106 TTGTTTAAAATACCGACCGTGTGATTGCTTCT

Stage107 GTAAATCGCGGTCATAGCCCCCTTCCGCCACC

Stage108 CTCAGAACTGGTGTAGATGGGCGCATCGTAAC

Stage109 GCGTTAAACCAATCCAAATAAGAAACGATTTT

Stage110 TTGCCATCATGTGAGTGAATAACCTAAATAAG

Stage111 CGTGCATCCTCAGGAGGTTTAGTAATTAGCGT

Stage112 TATTTATCTAAGAATAAACACCGGGTACATAA

Stage113 ATCAATATTTTTCATAATCAAAATTAGGTGTA

Stage114 TCACCGTATGCCAGTTTGAGGGGACGACGACA

Stage115 TTACTAGACAGTTACAAAATAAACAGCCATAT

Stage116 CCAGAGCCTTTTTTAATGGAAACAAATCATAA

Stage117 GTATCGGCTAAGTATAGCCCGGAACACCGGAA

Stage118 TAATTTGCAAAAGCCTGTTTAGTATTTCATTT

Stage119 GTACCTAGTGTCCAAGTCCT GAATTACCACCACCGGAACCGCCTGTCGAGAG

Stage120 GTACCTAGTGTCCAAGTCCT GGTTGATACTCAGGAAGATCGCACTCCAGCCA

Stage121 GTTATACAGCTAACGAGCGTCTTTCCAGAGCC

Stage122 GCCGCCACTAATTACATTTAACAATCATATGC

Stage123 GCTTTCCGCAGGCGGATAAGTGCCCCCTCAGA

Stage124 TTACCAACAATTCTTACCAGTATAATCAAGAA

Stage125 AACAAAATCCTCAGAACCGCCACCGGGTTTTG

Stage126 CTCAGTACGCACCGCTTCTGGTGCCGGAAACC

Stage127 GCTCAACACCAGCTACAATTTTATCCTGAATC

Stage128 CACCACCCAGATGATGAAACAAACAAGCCAAC

Stage129 AGGCAAAGAGGATTAGGATTAGCGCTCAGAGC

Stage130 TTTTGCACGTAGGGCTTAATTGAGTTACCTGA

Stage131 GCAAAAGATCAGAGCCGCCACCAGGAGACTCC

Stage132 TCAAGAGACGCCATTCGCCATTCAGGCTGCGC

Stage133 GTACCTAGTGTCCAAGTCCT TATTTAACGCCTTAAATCAAGATTAGTTGCTA

Stage134 GTACCTAGTGTCCAAGTCCT CCAGAGCCGAATTATTCATTTCAAAATCGCCA

Stage135 AACTGTTGAAAGTATTAAGAGGCTAACCACCA

Stage136 GTTTTGAAAACGCCAACATGTAATAAAATCGC

Stage137 GCAGAGGCGCCGCCAGCATTGACAATTATTCT

Stage138 GAAACATGGGAAGGGCGATCGGTGCGGGCCTC

Stage139 AGGCATTTAGCGAACCTCCCGACTTGCGGGAG

Stage140 AGGCAGGTTTGAATACCAAGTTACTTAGGCAG

Stage141 TTCGCTATGCCTATTTCGGAACCTGGAGGTTG

Stage142 GGCGTTTTTCGAGCCAGTAATAAGGATTCGCC

Stage143 TGATTGCTCAGACGATTGGCCTTGACAGTTAA

Stage144 TGCCCCCTTACGCCAGCTGGCGAAAGGGGGAT

Stage145 AAGTACCGTTATCCGGTATTCTAAGAACGCGA

Stage146 AAACAAATGGGAGAAACAATAACGAGAATATA

Stage147 GTGCTGCAAACAGTGCCCGTATAAATATTCAC

Stage148 TAGAAGGCACAAAAGGTAAAGTAACAGTACCT

Stage149 GTACCTAGTGTCCAAGTCCT TTTACATCAAATCCTCATTAAAGCGGTCAGTG

Stage150 GTACCTAGTGTCCAAGTCCT CCTTGAGTAGGCGATTAAGTTGGG

Stage151 AGACGACGGCCCAATAGCAAGCAAATCAGATA

Stage152 AAAGCGCAGATGAATATACAGTAATTCTGTCC

Stage153 GGGTTTTCTAATAAGTTTTAACGGCAGAATGG

Stage154 ATACGAGCTATCCGCTCACAATTCTAACGCCA

Stage155 GGAAACCTCGCTCACTGCCCGCTTCACACAAC

Stage156 ACGCTGGTTGAGAGAGTTGCAGCATCCAGTCG

Stage157 TGAGTGTTCAAAAGAATAGCCCGAAGCGGTCC

Stage158 CCCTAAAGGTGCCGTAAAGCACTAGATAGGGT

Stage159 GCTAGGGCGCTGGCAAAGCGAAAGGAGCGGGCAATCGGAA

Stage160 ATTACCGCACAATAAACAACATGTTTCAGGTT

Stage161 TAACGTCAGTCTCTGAATTTACCGTACAGGAG

Stage162 TGTACTGGCCAGTCACGACGTTGTTCCTGTGT

Stage163 GAAATTGTCGGAAGCATAAAGTGTCACATTAA

Stage164 TTGCGTTGGTCGTGCCAGCTGCATCTTCACCG

Stage165 CCTGGCCCTTGCCCCAGCAGGCGAAAAATCCC

Stage166 TTATAAATGTTCCAGTTTGGAACAGTTTTTTG

Stage167 GGGTCGAGGGAGCCCCCGATTTAGGAAAGGAA

Stage168 GGGAAGAAGTGTAGCGGTCACGCTGCGCGTAA

Stage169 TGCAGAACGTTTTTATTTTCATCGTAGGAATC

Stage170 AGCGTCATAGAAATTGCGTAGATTTCAGCTAA

Stage171 GGCCAGTGACATGGCTTTTGATGATTCCAGTA

Stage172 GTACCTAGTGTCCAAGTCCT GGGTGCCTCATGGTCATAGCTGTTAAAACGAC

Stage173 CGGCCAACAATGAGTGAGCTAACTAAAGCCTG

Stage174 TTTGATGGGCAACAGCTGATTGCCTAATGAAT

Stage175 CTATTAAATGGTTCCGAAATCGGCAAATCCTG

Stage176 GGGGAAAGCCATCACCCAAATCAAAGAGTCCA

Stage177 CCACCACACCGGCGAACGTGGCGAAGCTTGAC

Stage178 AGCAAGCCGCGCCTGTTTATCAACCGTAAAAC

Stage179 AGAAATAAACAAAGAAACCACCAGATTATCAT

Stage180 TTTGCGGACCAAGCTTGCATGCCTAGCTCGAA

Stage181 GTACCTAGTGTCCAAGTCCT TTCGTAATACCTTGCTGAACCTCAAAAAATCT

Stage182 AAAGCATCGCGCGGGGAGAGGCGGTTCACCAG

Stage183 TGAGACGGCAGTAATAAAAGGGACTCACCAGT

Stage184 CACACGACGAACGTGGACTCCAACTGGCCCAC

Stage185 TACGTGAATTATAATCAGTGAGGCATCCTGAG

Stage186 AAGTGTTTCCCGCCGCGCTTAATGCGCCGCTA

Stage187 AGTCCTGACAAGTACCGCACTCATCGAGAACA

Stage188 GAATTATCATCAAAATTATTTGCAAATAGATA

Stage189 ACTCTAGATAAAAGTTTGAGTAACAAGGAGCG

Stage190 ACCCTCAAGGATCCCCGGGTACCGGCAGGTCG

Stage191 TTGGGCGCGAGCCAGCAGCAAATGAATATCAA

Stage192 CAACAGAGCAGGGTGGTTTTTCTTTTTGCGTA

Stage193 CTACTGCGACATCTTATCTA GCGAAAAAATTTACATTGGCAGATATTCTGGC

Stage194 AAAAGAGTCCGTCTATCAGGGCGAGTCAAAGG

Stage195 CAGGGCGCGGAACGGTACGCCAGACACCGAGT

Stage196 TATTAAACACAAGAAAAATAATATGGTTAGAA

Stage197 CCTACCATATCATATTCCTGATTACGAACGTT

Stage198 ATTAATTTCGTCAATAGATAATACACTAATAG

Stage199 ATTAGAGCTCAATATCTGGTCAGTAACAGTGC

Stage200 CACGCTGAACATCGCCATTAAAAAAACTGATA

Stage201 GCCCTAAAATAGAACCCTTCTGACTCGTCTGA

Stage202 AATGGATTGGTAATATCCAGAACAAACTATCG

Stage203 GCCTTGCTCTGTCCATCACGCAAAAAAGGGAT

Stage204 TTTAGACAGTACTATGGTTGCTTTGACGAGCA

Stage205 AATTTACGTTTCCTTATCATTCCAAGAACGGG

Stage206 TGGCAATTTTCTGAATAATGGAAGCCCATCCT

Stage207 ATTTAGAATATTAAATCCTTTGCCTCAGATGA

Stage208 CAACAGTTTTTAGGAGCACTAACAATTTGAGG

Stage209 GAACCACCTATTAACACCGCCTGCTGGCAAAT

Stage210 GTAAGAATTAGTCTTTAATGCGCGTACCGAAC

Stage211 GCCAGCCAACATTTTGACGCTCAACTGAAAGC

Stage212 TGTAGCAATGAGTAGAAGAACTCAATATTACC

Stage213 CGTATAACAAACAGGAGGCCGATTTTAACCGT

Stage214 TCAATAATCGGCTGTCAGCATGTAGAAACCAATTTTATTGTTTG

Stage215 GATTATACCATCAATATAATCCTGTTTTACAATTCG

Stage216 ACAACTCGGTATTAGACTTTACAATTTTGGTTATCT

Stage217 AAAATATCGAAAGGAATTGAGGAATTTTGAGGTGAG

Stage218 GCGGTCAGAGCAGAAGATAAAACATTTTATTTTTGA

Stage219 ATGGCTATACGTGGCACAGACAATTTTTGCTCATGG

Stage220 AAATACCTTTGCAACAGGAAAAACTTTTAATAACAT

Stage221 CACTTGCCTACTTCTTTGATTAGTTTTTAATCAGAG

Stage222 CGGGAGCTGTGCTTTCCTCGTTAG

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