Biomod/2012/UTokyo/UT-Komaba/Experiment/Original Origami: Difference between revisions

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DNA origami is needed to make the DNA tablet because the main body of the tablet is based on DNA origami.
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.
However, the best buffer conditions for a DNA origami and the bistable system are not same, so we searched the optimal working conditions to combine them.
We also test if we can observe DNA origami in mixed liquid because the bistable system contains three kinds of enzymes and some proteins.
We also tested if we can observe DNA origami in mixed buffers because the bistable system contains three kinds of enzymes and some proteins that may damage the origami.
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.  
The purpose of the experiment is to find conditions compatible with both the origami and the bistable system and to make sure that the enzymes of the bistable system do not destroy the DNA origami. We also wanted to check that we can observe by AFM the origami while in a mix that contains proteins.
The DNA origami we used consists of m13 and a set of 216 staples, which was designed by Shelley F. J. Wickham et al[[Biomod/2012/UTokyo/UT-Komaba/Supplementary#References|[1]]].
The merit of this origami is that it is as flat as possible, and it is thus easier to observe its surface by AFM.


==Task==
==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(BST, ttRecJ and NBI). They may destroy DNA origami.
* DNA origami needs Mg+ to shape well, while Mg+ can prevent enzymes from work correctly.


In experiments below, we tried to find out the compromise of condition between DNA Origami and the bistable system.
===Compatibility with the Bistable System===
 
In order to combine DNA origami with the bistable system, we needed to overcome the difference in their best conditions.
* The bistable system uses some enzymes (a polymerase -Bst large Fragment-, an exonuclease -ttRecJ- and a nicking enzyme -NBI) which may destroy DNA origami. The polymerase could extend the 3'end of the staples strands along the scaffold, starting form the nicks; the exonuclease could slowly degrade the staple strands.
* The bistable works at higher temperature, so this may also impact the stability of the structure.
* The bistable system also uses BSA. It can prevent origami to stick to mica, and we may not get good images by AFM.
 
In experiments below, we tried to find out the compromise of conditions between DNA origami and the bistable system which enables both the bistable system and DNA origami to work properly.
 
The direct link to the result of the experiments about compability with the bistable system is [[Biomod/2012/UTokyo/UT-Komaba/Experiment/Compatibility_with_Bistable|here]].


==Experiment==
==Experiment==
Line 23: Line 30:
===September 12th===
===September 12th===


We made a solution of DNA origami.
We made a solution of the DNA origami.
We put M13 and the 216 kinds of staple strands in tubes and made them anneal for about an hour in a PCR machine.
We made three tubes of origami solution whose difference is the concentration of staple strands.


(µL)
[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#September_12th|More Information]]
{|
!
! 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===
===September 13th===


We observed DNA origami which we made September 12th by AFM.
We observed DNA origami which we made on September 12th by AFM.
We used the AFM at Suyama Lab in Komaba I Campus.
We could not observe the middle-concentrated solution of origami.


 
*AFM Image of Low-Concentrated Solution of Origami
*AFM image of A
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_origami-AFM-1.jpg|300px]]              [[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_origami-AFM-2.jpg|300px]]
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_origami-AFM-1.jpg|300px]]              [[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_origami-AFM-2.jpg|300px]]




*AFM image of C
*AFM Image of High-Concentrated Solution of Origami
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_origami-AFM-3.jpg|300px]]
[[Image:Biomod_2012_UTokyo_UT-Komaba_Experiment_origami-AFM-3.jpg|300px]]


As you can see from the picture above, in both solutions, no origami was well structured.
Therefore, we decided to do the same experiment again without keeping them in the freezer for a day.


===September 19th===
[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#September_13th|More Information]]
 
*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
|}


===September 14th===


*DNA Origami with Enzymes
We made the DNA origami solution again.
We steeply lowered the concentration of staple strands.
Also, in the experiment, the origami was not kept in the freezer but observed by AFM just after it was annealed .


We use enzymes in bistable system, therefore we make sure that the enzymes do not destroy the DNA origami.
[[Image:BIOMOD-2012-UTokyo-UTKomaba-plainorigami.jpg]]
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)
As you can see the picture above, the origami was well structured.
{|
!
!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
|}


[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#September_14th|More Information]]


*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)
__NOEDITSECTION__
{|
!
!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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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. However, the best buffer conditions for a DNA origami and the bistable system are not same, so we searched the optimal working conditions to combine them. We also tested if we can observe DNA origami in mixed buffers because the bistable system contains three kinds of enzymes and some proteins that may damage the origami. The purpose of the experiment is to find conditions compatible with both the origami and the bistable system and to make sure that the enzymes of the bistable system do not destroy the DNA origami. We also wanted to check that we can observe by AFM the origami while in a mix that contains proteins. The DNA origami we used consists of m13 and a set of 216 staples, which was designed by Shelley F. J. Wickham et al[1]. The merit of this origami is that it is as flat as possible, and it is thus easier to observe its surface by AFM.

Mission

Compatibility with the Bistable System

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

  • The bistable system uses some enzymes (a polymerase -Bst large Fragment-, an exonuclease -ttRecJ- and a nicking enzyme -NBI) which may destroy DNA origami. The polymerase could extend the 3'end of the staples strands along the scaffold, starting form the nicks; the exonuclease could slowly degrade the staple strands.
  • The bistable works at higher temperature, so this may also impact the stability of the structure.
  • The bistable system also uses BSA. It can prevent origami to stick to mica, and we may not get good images by AFM.

In experiments below, we tried to find out the compromise of conditions between DNA origami and the bistable system which enables both the bistable system and DNA origami to work properly.

The direct link to the result of the experiments about compability with the bistable system is here.

Experiment

September 12th

We made a solution of the DNA origami. We put M13 and the 216 kinds of staple strands in tubes and made them anneal for about an hour in a PCR machine. We made three tubes of origami solution whose difference is the concentration of staple strands.

More Information

September 13th

We observed DNA origami which we made on September 12th by AFM. We used the AFM at Suyama Lab in Komaba I Campus. We could not observe the middle-concentrated solution of origami.

  • AFM Image of Low-Concentrated Solution of Origami


  • AFM Image of High-Concentrated Solution of Origami

As you can see from the picture above, in both solutions, no origami was well structured. Therefore, we decided to do the same experiment again without keeping them in the freezer for a day.

More Information


September 14th

We made the DNA origami solution again. We steeply lowered the concentration of staple strands. Also, in the experiment, the origami was not kept in the freezer but observed by AFM just after it was annealed .

As you can see the picture above, the origami was well structured.

More Information


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