We divided wet experiment into two parts.
First, we experimented resistance part in A/D converter. Next, we experimented logic gate part in A/D converter.

Figure 1: Dividing experiment parts in A/D converter

1. Resistance part in A/D converter

Figure 2: Our idea of how to use Universal Strand Generator

Figure 3: Difference between Team Sendai and Team YOKABIO

We obtained cooperation from Team Sendai and experimented Universal Strand Generator(enzyme free device) which was experimented by Team Sendai in biomod2014.
We realized resistance part in A/D converter by using Universal Strand Generator to release outputs at different concentration. Each DNA sequence is shown below.

Table 1: DNA sequences we used in resistance part of wet experiment

We wanted to obtain two outputs in this experiment. One is released from step1 and the other is released from step2. The simplified reaction process is shown in Figure 4.

Figure 4: Simplified reaction process

Methods

Step 1: We generated double-strand DNA. Each DNA was annealed by using thermal cycler (from 95 to 20℃, -1℃/min). The detail of annealing is shown below.

Table 2: The detail of annealing (* means complementary strand)

Step 2: First, we put transducerA, transducerB, gateA, gateB, fuelA, fuelB and trigger in tube. After that, we put input in tube. When we used inputAB as input, the outputA corresponds to output1 in Figure 4. And, when we used inputBA as input, the outputA corresponds to output2 in Figure 4. In this time, we divided experiment into two patterns (pattern1 and pattern2), and measured fluorescence intensity by using spectrofluorometer(FP6200, JASCO) to confirm whether the outputA is released. The outputA includes fluorescent molecule (6-FAM).
If you want to see detail of reaction process and role of fluorescence molecule, please see the web site of Team Sendai in biomod2014. The URL is shown below.

BIOMOD2014 Team Sendai Wiki
http://teamsendai2014.github.io/protocol.html#Electrophoresis

Pattern 1: We used inputAB
OutputA is released before releasing outputB. And we set transducerA concentration to 80nM to release outputA concentration of 80nM.
Following table shows the DNA we used in pattern1 of wet experiment.

Table 3: Used DNA and its concentration in pattern1

We reacted DNA showing above and measured fluorescence intensity of outputA by using spectrofluorometer(FP6200,JASCO). Figure 5 shows fluorescence intensity of outputA at wavelength 518nm. We could confirm outputA is released in Figure 5. So we can say step1’s reaction of Figure 4 is functioned.

Figure 5: Fluorescence intensity of outputA when we used InputAB. TransducerA concentration is 80nM.

Pattern 2: We used inputBA
We put above DNA and measured fluorescence intensity of outputA by using spectrofluorometer(FP6200,JASCO).
Figure 6 shows the fluorescence intensity of outputA at wavelength 518nm. We could confirm that outputA is released in Figure6. Therefore we could see that step2’s reaction of Figure 4 is achieved.

Figure 6: Fluorescence intensity of outputA when we used InputBA. TransducerA concentration is 120nM.

In conclusion, we could confirm that fluorescence intensity of pattern2 is higher than pattern. This is because we set that pattern1’s outputA concentration is 80nM and pattern2’s outputA is 120nM.
Moreover, we could confirm that pattern2 needs reaction time more than pattern1. This is because in pattern2, outputA in step2 is released after outputB in step1 is released. For this reason, we could generate two outputs having different concentration by using Universal Strand Generator.

2. Logic gate part in A/ D converter

We experimented Threshold gate and NOT gate in A/D converter. To confirm whether output is released, we ran agarose gel electrophoresis.

Figure 7: Expectation of threshold gate and not gate reaction

Materials and Methods

Step 1: Design DNA sequences
We designed DNA sequences with NUPACK.
We considered minimum free energy of each reaction and designed DNA sequences for DNA strand displacement reaction easily.

Table 5: Threshold gate sequences

Table 6: NOT gate sequences

Step 2: DNA preparation
We ordered all DNAs of liquid state and those concentrations are 100μM to Wako Pure Chemical Industries, Ltd. We diluted DNAs with following TE buffer and regulated the DNA concentration as Table8. Then, DNA are Stirring with vortex and centrifugal machine.

Table 7: TE buffer Composition

Table 8: Each element concentration

Figure 8: Incubator (ASTEC)

Figure 9: Electrophoresis chamber(Mupid)

Step 3: Annealing
Threshold and Gate:output were formed by cooling with incubator. (from 95 to 20℃, -2℃/min)
Step 4: Making 5% agarose gel
We added agarose(5mg) and TBE buffer(95ml) and heated them in the microwave oven until agarose and TBE buffer completely dissolved. After the agarose had cooled, poured the agarose in the holder which was set the comb.Agarose hardened completely, removed the comb and moved agarose to chamber. We added TBE buffer to the chamber.

Table 9: TBE buffer Composition

Step 5: Toehold exchange reaction progression
We put all DNA into the tube and mixed them at normal temperature.

Step 6: Running the agarose gel electrophoresis
We added mixed DNA (5μl) and 6× Low Molecular Weight DNA Ladder(1μl). Next, we applied mixed solution (6μl) to each gel-well, and ran electrophoretic device at 100V, for 30mins.
When we finished electrophoresis, we moved gel to the container that is added GelRed (1μl). We shook the container with seesaw shaker. Putting elements in each lane as follow, and we verified output’s difference whether the input was high or low.

・Threshold gate
     1Lane Input(high),Threshold
     2Lane Input(low), Threshold
     3Lane Input(high), Threshold, Gate:output, Fuel
     4Lane Input(low), Threshold, Gate:output, Fuel

・NOT gate
     5Lane Input(high)
     6Lane Input(low)
     7Lane Input(high), Inverter, Threshold, Gate:output, Fuel
     8Lane Input(low), Inverter, Threshold, Gate:output, Fuel

Step 7: Observing the band with gel imager.

Figure 10: Gel imager (NIPPON Genetics)

Result
Unfortunately, we could not obtained the desired results with electrophoresis in which some experimental conditions might not be adequate although the basic experimental conditions had determined in preliminary experiments.

Reference
・Team Sendai(2014) Materials and Methods. Available
at : http://teamsendai2014.github.io/protocol.html (Accessed 14 October 2015 )