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Team:MIT/NOTGate
From 2012.igem.org
Our NOT Gate Design
Design
The NOT gate (signal inverter) design we eventually used comprises of multiple double- and single-stranded species. The most important of these are NOT gate (A), output (B) and buffer (C) complexes.
There are two cases to consider: first the case where there is no input present (in digital terms, the input is 0), and second the case where there is a saturating level of input (digitally represented as a 1). We aim to convert the '0' input into a '1' output and the '1' into a '0' - the basic operation of a NOT gate. Refer to the diagram below throughout the explanation.
Case 1: No input present -> high signal
Here, the output strand (B) reversibly reacts with the NOT gate (A). The reaction is reversible because the output strand only displaces the green section on the NOT gate, but not further, meaning that the reaction could go back as well. This reversible reaction allows the output strand to react to a downstream buffer (C) and amplifier (D) to produce a high signal (a new active nucleic acid strand is released).
Case 2: Input present -> low signal
Here, the input strand and output strand both react with the NOT gate. The input strand displaces up to the pink region and the output strand displaces up to the green region. This is irreversible, as once strand displacement happens from both sides, the complex separates into two double-stranded species. There is no toehold to initiate the reverse reaction, so the output strand is "stuck". The output therefore cannot be buffered or amplified to produce a signal: no new active nucleic acid strand is released.
Diagram of our NOT gate. See the paragraph above for a more detailed description.
To get signal amplification we use a fuel molecule (D). Once the output (B) reacts with the buffer (C), we get two complexes: the single-stranded signal and a double-stranded intermediate (see Case 1 above). This intermediate has a green toehold and a pink hybridization domain, which matches the toehold and domain on the fuel molecule, allowing the the two react. The reaction results in a fuel:gate waste complex (not shown) and a free output strand (B), which is now able to react with another buffer. Signal amplification results from the ability of one strand of B to produce multiple signal strands.
Initial Simulations
We initially designed three NOT gates, each using different components. Click on the pictures below to see a bigger reaction diagram.
Design 1:
Design 2:
Design 3:
We simulated each of these designs in Visual DSD, a program designed to simulate strand displacement reactions. Based on our simulation results, we decided to design the sequence, order, and test the third design.
A sample of Visual DSD code. You can download the actual code for this NOT sensor design here. Copy the code into Visual DSD to execute it.
A reaction diagram generated by Visual DSD, based on the above code.
The results of our simulation. The final design is compared against an earlier design. Amount of fluorescence indicates the fluorescence from a downstream reporter.
In Vitro Results for Our NOT Gate
To test the functionality of the NOT gate as expressed by its transfer function (relating the input levels to output signal strengths) we performed in vitro studies with DNA as the nucleic acid for strand displacement. The output signal strand was fed to a reporter complex. This reporter, when triggered by a signal, will fluoresce in the red channel. Our experiments measured these fluorescence levels for various amounts of initial input levels using a plate reader.
Optimization
The original in vitro test showed a result that did not match our simulated transfer function.
Initial in vitro studies of the NOT gate in DNA. All components at equal concentrations, with 1x=1 pmol. We vary the amount of input strand added and measure the resulting fluorescence output levels which we then normalize to the maximum.
From the simulations, we knew that by increasing the concentration of the dynamic gate (complex A), we would get a more digital signal. We implemented this strategy by increasing the relative concentration of strand A by various amounts.
Improved in vitro studies of the NOT gate in DNA. Again, we vary the amount of input strand added and measure the resulting fluorescence output levels which we then normalize to the maximum.
Looking at the low dip in the middle concentration ranges and the rise afterwards, we determined that the cooperative hybridization was not working at the levels we were using. To test this, we raised the absolute concentration of all the parts of the NOT gate and kept the high relative concentration of complex A. This resulted in a transfer function that very nearly matched our simulation and gave us NOT gate behavior.
Final in vitro studies of the NOT gate in DNA. Concentration of strand A is 1.4X, where X = 20pmol. We overlay the experimental results (blue) to our simulation results (red). We observe that the NOT gate is producing the desired transfer function (high output signal as measured by fluorescence when the input strand is in low amount and low output signal when the input strand is in high amount).
