Team Kyutech

Kyushu Institute of Technology　Nakakuki lab

Address

Kyushu Institute of Technology 680-4 Kawazu, Iizuka, Fukuoka, Japan

3. Experiment

The final goal in the Molecular Governor project is to establish a feedback control method for regulating a function of a biochemical reaction system. To this end, the most important device is the membrane with a desired transmission rate, and the most important technology is how to create such a membrane. In BIOMOD2014, we focus on the manufacturing of the membrane. Because we proposed the design theory in the Design section, in this section we try to validate it experimentally.

3.1 Break through

You may consider that it would be difficult to make such a membrane as shown in Fig. 1 even if the design theory is correct. The difficulty comes from the following matters.

● Permeable membrane that a DNA strand can pass through with desired transmission rate.

● How to integrate the membrane in an existing system

At the moment, many kinds of permeable membrane are available. To create one, a technique to precisely make small pores on a liposome may be highlighted although, unfortunately, it is still a difficult work. In addition, although such a “sheet-type” permeable membrane (Fig. 1) would be a wonderful material, to handle or integrate it in an existing system would be a troublesome problem. Our breakthrough is to provide a concept for making a permeable membrane quite easier.

Fig.1 Typical realization of permeable membrane

3.1.1 Problem

● It’s difficult to exactly make nanoscale pores on a sheet.

● It’s difficult to handle or integrate it in an existing system.

Now, we propose the following solution. A permeable membrane is created by sticking poles, which would be made by DNA origami, on an impermeable sheet. Each pole has many nanoscale pores that provide a permeable property. In BIOMOD2014, to confirm the principle we employed a ceramic membrane filter (NGK Insulators, ltd.) as a pole in which the concept, the three-dimensional structure, and the flux flow of solute are exactly the same with our idea. Fig.3 illustrates the flux flow of solute and solvent.

Fig.2 Ceramic membrane filter (NGK Insulators, Itd.)

3.1.2 Solution

● The three-dimensional structure and the flux flow of solute and solvent are exactly the same with our idea.

● A pole-style filter with many nanoscale pores.

● It is possible to integrate it into an existing system by stick it on an impermeable plane.

Fig.3 flux flow of solute and solvent

3.2 Materials and Methods

3.2.1 Materials

We summarize the materials in our experiments. A water tank was originally made by us, and has two rooms separated by an impermeable divider. A ceramic membrane (NGK Insulators, Ltd.) is attached to pass through the divider, therefore, two cross-border rooms are connected with the ceramic membrane. In this experiments, we employed a DNA oligonucleotide (synthesized by Takara Bio Inc.) as a solute and pure water as a solvent. A spectrophotometer was employed to quantitatively measure the abundance of DNA strand. All calculation was performed with Matlab (The MathWorks, Inc.). Each materials are shown as follows:

● Synthetic DNA oligonucleotide

Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure(sequence). In this time, we use DNA oligonucleotide has below characters.

Form: : freeze-dried product
Length : 20base
Sequence : 5’-ACCTCTCATCTATCACCACC-3'
Molecular weight : 5918.0

● Water tank(handled by us) and ceramic membrane(NGK Insulators, Ltd.)

● Characters of water tank is below    Radius : 10[mm]
   Length : φ200[mm]
   Material : acrylic plastic

● Characters of water tank is below.    Meterial : Titania
   Pore size : φ100 [nm]
   Channel : 19
   Length : φ15[cm]
   Surface area : φ3[mm]

● Spectrophotometer

Spectrophotometer has enhanced design features enabling rapid calculations of various parameters based upon the sequence of oligonucleotide. Selection of the appropriate mode and calculation key gives the user an output of melting temperature, absorbance, concentration and molecular weight based upon the sequence of oligonucleotide.

3.2.2 Method

The procedure of the experiment is illustrated in Fig. 4 in which DNA strands were administrated into the left room, and then a concentration of the DNA strand in the right room is measured by the spectrophotometer at regular intervals to make a time-course plot.

Fig.4 overall view of experiment

3.2.3 Procedure of experiment

Before we mention about procedure, we simulate concentration change by reference to parameter of DNA and membrane that we use in experiment.

(1)Put water in the water tank.

(2)Administrate the DNA oligonucleotide into the left room.

(3)Retrieve a specific amount of solution from the right room at regular intervals (Fig. 5).

(4)Quantify the concentration by using spectrophotometer.

(5)Compare the concentration change between simulation result and actual measurement.

Fig.5 Retrieve a specific amount of solution at regular intervals

3.3 Result

Fig.6 shows two kind of time-course from the simulation with parameter of DNA and membrane that we use in experiment and the result with parameter of actual measurement in experiment. The both of the two lines show the concentration of DNA after it passes through a membrane.

Fig.6 Comparison of concentration change (blue, Matrab simulation; red, actual measurement)

Fig.6 illustrates that the concentration change of the theoretical value and actual measurement value after passes through　tend to increase until 45 seconds. Therefore, the result of our experiment provides evidence that DNA can pass through the membrane. After 45 seconds, the actual concentration of DNA showed unstable concentration change.

3.4 Discussion

In conclusion, we found that nano-scale DNA can pass through a ceramic membrane filter. However, in this experiment, the variations of concentration change were observed. Therefore we discuss the reason as follows.

● It takes time until concentration in solution becomes constant.
● We didn’t consider the surface tension of membrane.

To solve them, we discuss that we should use solvent as low viscosity as possible.

3.5 Future perspective

We plan on examining as follows.

● DNA strand displacement reaction is integral control by using membrane.
● Realize membrane by DNA origami.

If our technology is established, we can realize a integral control with membrane of DNA origami and we can open the door of breakthrough appreciation in the future.