Biomod/2012/UTokyo/UT-Komaba/Experiment/Original Origami

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<div id="title"><img src="http://openwetware.org/images/4/47/Biomod_2012_UTokyo_UT-Komaba_Top.png" alt="DNA tablet" width="800" height="120" onClick="this.src='http://openwetware.org/images/7/7d/BIOMOD_2012_UTokyo_UT-Komaba_title-animation.gif'"/></div>

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Concept

Original Origami Concept
Original Origami Concept


DNA origami is needed to make the DNA tablet because the main body of the tablet is based on DNA origami. We combine the bistable system and DNA origami whose best conditions are not same, so that we searched the best condition of mixed liquid. We also test if we can observe DNA origami in mixed liquid because the bistable system contains three kinds of enzymes and some proteins. The purpose of the experiment is to find the best condition of mixed liquid and to make sure that enzymes of the bistable system do not destroy DNA origami and that we can observe it in liquid which contains proteins by AFM.

Mission

Compatibility with bistable system

In order to combine DNA Origami with the bistable system, we needed to overcome difference in their best conditions.

  • The bistable system uses some enzymes( apolyerase -Bst large Fragment-, an exonuclease -ttRecJ- and a nicking enzyme -NBI). They may destroy DNA origami.
  • DNA origami needs Mg2+ to shape well, while Mg2+ also strongly affects the activity of enzymes and may prevent them from working properly.

In experiments below, we tried to find out the compromise of condition between DNA Origami and the bistable system, so as to both the bistable system and DNA Origami would work correctly.

Experiment

Scaffold and staple strands

The Origami we used is consists of m13 and set of 216 staples, taken directly from a previously published design.


September 12th

We made a solution of DNA origami.

(µL)

M13 Staple Mix TAE 10x Mg2+ mQ Total Amount
A 3.00 7.20 1.80 2.25 3.75 20.00
B 3.00 3.60 1.80 2.25 7.35 20.00
C 3.00 10.80 1.80 2.25 0.15 20.00

September 13th

We observed DNA origami which we made September 12th by AFM.

  • AFM image of A


  • AFM image of C


September 14th

We made DNA origami solution again.

(µL)

M13 staple mix Mg TAE mQ total amount
1.66 2.16 2.5 2 11.68 20

We observed again, and the origami was successfully structured.


September 19th

  • DNA Origami

We prepared two types of DNA origami solution. The purpose of the experiment was to compare the different result from the different concentration of the solutions.

(µL)

Staple Mix M13 TAE 10x Mg2+ mQ Total Amount
A 8.00 3.33 2.00 0.25 6.42 20.00
B 12.00 5.00 2.00 0.25 0.75 20.00


  • DNA Origami with Enzymes

We use enzymes in bistable system, therefore we make sure that the enzymes do not destroy the DNA origami. We put nickase, polymerase and exnuclease with DNA origami and left them in the PCR. The concentration of original DNA origami was 10x and 15x.

(µL)

BST NBI tt-RecJ DTT BSA Smix DNA origami mQ Total Amount
DNA origami 10x 0.20 0.80 0.30 0.20 0.20 5.00 10.00 3.30 20.00
DNA origami 15x 0.20 0.80 0.30 0.20 0.20 5.00 10.00 3.30 20.00


  • DNA Origami with BSA

We are also worried if BSA interrupt the AFM and we can not get clear images of DNA origami. Therefore, we prepare the solution for September 20th. The concentration of original DNA origami was 10x and 15x.

(µL)

DNA origami Smix 4x BSA mQ Total Amount
DNA origami 10x 2.00 5.00 2.00 11.00 20.00
DNA origami 15x 2.00 5.00 2.00 11.00 20.00
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