Biomod/2012/UTokyo/UT-Komaba/Experiment/Compatibility with Bistable

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(Compatibility with the Bistable System)
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[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#September_20th|More Information]]
[[Biomod/2012/UTokyo/UT-Komaba/Experiment/Lab_Notes#September_20th|More Information]]

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DNA tablet


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.


September 19th

  • Normal DNA Origami

We prepared two types of DNA origami solution. The purpose of the experiment was to compare the different results from the different concentrations of the solution. Both concentrations of staple strands were as high as we did in September 12th.

  • DNA Origami with Enzymes

We use enzymes in the 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 stored them at the working temperature of the bistable. We used the DNA origami solution made on September 19th, enzymes and some essential chemicals

  • DNA Origami with BSA

We were also worried if BSA interrupt the AFM and we can not get clear images of DNA origami. Therefore, we prepared a mix for September 20th. We put DNA origami solution made on September 19th, BSA and some essential chemicals.

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September 20th

We observed three types of DNA origami made on September 19th. The three types is normal DNA origami, DNA origami with BSA and DNA origami with enzymes.

  • Normal DNA Origami

We observed the origami by AFM, but the structures of both DNA origami were destroyed as you can see from the picture below. We suspect that it is due to keeping the origami in the freezer over night.

  • DNA Origami with BSA

We need BSA for the bistable sytem so that we had to make sure that we can see the DNA structure when there are BSA in the liquid of the origami. Before we observed the origami with BSA by AFM, we rinsed 5μL of the origami liquid with 40μL of buffer for 4 times. Therefore, as you can see from the picture below, BSA did not disturb the observation of AFM, we can get the clear picture of DNA structure.

  • DNA Origami with Enzymes

In the bistable system, we use three kinds of enzymes so that we had to make sure that the enzymes do not destroy the origami. As the result, we could see the DNA structure clearly, which proved that the enzymes do not destroy the DNA origami. Moreover, we observed the well-done origami structure in this experiment.

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