Team:OUC-China/Proof Of Concept
1. Overview
This year, OUC-China is inspired by biocomputer. We consider biocomputer should have special functions which emphasis on biological characteristic and would be complementary with electronic computers eventually. Since the basic functional unit of a computer is implemented through logic gates, we decided to start with that. We hope to reflect on the application value of our project from the two aspects of calculation and detection. In fact, the experiments we have completed have proved that it is feasible to implement calculation and detection using logic gates.
2. Calculation
We construct the half-adder and the subtractor with the AND, XOR and NIMPLY gates. These logic gates have been successfully tested in previous experiments. In the following paragraphs, we will elaborate further on our validation.
2.1 Half-adder
We used the AND gate and XOR gate to make up the half-adder. A half adder is a type of adder, an electronic circuit that performs the addition of numbers. The half adder is able to add two single binary digits and provide the output plus a carrying value. It has two inputs, called A and B, and two outputs S (sum) and C (carry).
When there is only one input, the reporter of the AND gate is not expressed, and the reporter of the XOR gate is expressed. When there are two inputs, the reporter of AND gate is expressed, and the reporter of XOR gate is not expressed.
Figure 1. Half-adder circuit diagram
2.1.1 XOR Gate
The XOR is inspired by the NIMPLY gate, consisting of a toehold switch and two triggers (figure 2). The trigger’s core sequence is the same and at the triggers’ both ends there are the nucleotide-binding domains. When input one of these triggers, the switch can turn on. Moreover, when input these two triggers simultaneously, they can pair together and form a ring in the middle. As a result, the switch will still be in the OFF state.
Figure 2. Demonstration of XOR gate
The nucleotide-binding domains were designed using NUPACK. And the core sequences of trigger originate from previous literature (figure 3). In our results, the output of XOR gate is consistent with our expectation after the inducer is input according to the truth table (figure 4).
Figure 3. The secondary structure of the pairing two triggers analyzed using NUPACK.
Figure 4. Two-input toehold XOR gate
The left figure shows that when INPUT A and INPUT B are both 0 or 1, the fluorescence of GFP is low. And the fluorescence intensity of GFP was high in the other two groups. This corresponds to the situation described in the truth table on the right. INPUT A=1 means that aTc (0.25 mg/mL) is added, INPUT B=1 means that HSL (0.1 mg/mL) is added. Error bar: SD (n=9).
2.1.2 AND Gate
AND gate constructed from two input RNAs that bind to yield a complete trigger RNA (figure 5). When either input RNA is expressed, it is incapable of activating the switch because neither trigger sub-sequence alone can unwind the repressing hairpin. The toehold switch can only be turned on when the two input RNA species hybridize and form a complete trigger sequence. In our results, the output of AND gate is consistent with our expectation after the inducer is input according to the truth table (figure 6).
Figure 5. Demonstration of AND gate
Figure 6. Two-input toehold AND gate
The left figure shows that when INPUT A=1, INPUT B=1, the fluorescence of GFP is high. And the fluorescence intensity of GFP was low in the other three groups. This corresponds to the situation described in the truth table on the right. INPUT A=1 means that aTc (0.25 mg/mL) is added, INPUT B=1 means that HSL (0.1 mg/mL) is added. Error bar: SD (n=9).
2.2 Subtractor
We used the NIMPLY gate to make up the subtractor. We use inducers to turn on trigger expression, use inducer concentration to represent the input trigger concentration, and use fluorescent protein expression levels to react and output results. When two triggers are expressed at the same time, trigger S1 and trigger S2 will combine to form an RNA double strand that cannot be turned on by RNA Switch. Only when trigger S2 is expressed, the downstream gene expression of RNA Switch will be turned on. This subtracter can simulate S2-S1=Ps, and Ps is the fluorescence intensity of the reporter gene.
Figure 7. Abstractions of subtractor
2.2.1 Design of NIMPLY Gate
In the NIMPLY gate, a deactivating RNA (INPUT A) uses direct hybridization or strand displacement to abolish trigger RNA (input B) activity. So when only the correct trigger RNA is expressed, the switch can be turned on (figure 8).
Figure 8. Demonstration of NIMPLY gate
However, we only find one NIMPLY gate sequences in the literature. In order to achieve the subtractor that can calculate different concentration range, we designed NIMPLY upon that sequence.
The NIMPLY gate consists of a toehold switch and two input triggers that can pair together. The toehold and the triggers’ core sequences we used originated from the previous literature. Then we added the nucleotide-binding domains at both ends of the trigger’s core sequence. This sequence is named “trigger1”. The other one was designed for completely complementary pairing with trigger1 and not pairing with switch sequence. These sequences were designed using NUPACK (figure 9) and were further screened using RNAfold and RNAstructure.
Figure 9. The secondary structure of the pairing two triggers analyzed using NUPACK.
2.2.2 Proof of NIMPLY Gate
In our results, the output of NIMPLY gate is consistent with our expectation after the inducer is input according to the truth table (figure 10).
Figure 10. Two-input 3WJ repressor NIMPLY gate
The left figure shows that when INPUT A=0, INPUT B=1, the fluorescence of GFP is high. And the fluorescence intensity of GFP was low in the other three groups. This corresponds to the situation described in the truth table on the right. INPUT A=1 means that aTc (0.25 mg/mL) is added, INPUT B=1 means that HSL (0.1 mg/mL) is added. Error bar: SD (n=9).
3. Detection
Using biological computers for detection is another goal we want to achieve. We will take virus detection as an example to illustrate that our logic gate can realize detection function. The use of logic gates can reduce false negative and false positive of virus tests and also makes it possible to detect multiple viruses simultaneously.
3.1 Reduce False Negative
A false negative in a test can often cause people to ignore the danger. For viruses that often produce new mutated individuals, testing for a single fragment is likely to result in omissions. Using OR gate (figure 11) can effectively solve this problem, we can select a specific segment from the original virus and the novel virus for testing, as long as any segment exists, the reporter gene can be expressed. In our results, the output of OR gate is consistent with our expectation after the inducer is input according to the truth table (figure 12).
Figure 11. Demonstration of OR gate
Figure 12. Two-input toehold OR gate
The left figure shows that when INPUT A=0, INPUT B=0, the fluorescence of GFP is low. And the fluorescence intensity of GFP was high in the other three groups. This corresponds to the situation described in the truth table on the right. INPUT A=1 means that aTc (0.25 mg/mL) is added, INPUT B=1 means that HSL (0.1 mg/mL) is added. Error bar: SD (n=9).
3.2 Reduce False Positive
False positives are another major challenge in virus testing. Many homologous viruses have a large number of highly similar or even identical conserved fragments. We found that the use of the AND gate containing an inhibiting hairpin increased the specificity of the detection (figure 13). We designed the binding domain to inhibit the hairpin based on the same sequence of the homologous virus, and designed the second hairpin based on the specific fragment of the target virus we wanted to detect.
Figure 13. Demonstration of AND gate
3.3 Multi-virus Detection
In complex environments, there is often more than one virus. For example, Noroviruses (NoV), Hepatitis A (HAV), Coronavirus, and other pathogenic viruses often exist in the hospital environment. This can easily lead to alimentary infection, respiratory infections, operation slices infection, and other hospital infections.
The programmability of RNA-based regulatory elements is based on the sequence complementary pairing, which gives the biosensor good specificity. By designing particular regulatory elements, we could detect multiple viruses simultaneously in an orthogonal way. Furthermore, as mentioned above, by combining different types of logic gates, we can improve the biosensor performance by avoiding false-negative and false-positive reports. According to the expression of different logic-gate reporters, we can determine the presence of various viruses.
4. Supplementary Validation (Electrophoresis Result of Expression Vector)
In order to verify the two-plasmid system, we introduced into E. coli BL21 Star (DE3), we designed the upstream and downstream primers of the inserted fragment and used these primers for colony PCR. The results of gel electrophoresis after colony PCR are shown in the figure (figure 14) below.
Figure 14. The results of gel electrophoresis.
From left to right respectively: lane 1: AND gate, lane 2: OR gate, lane 3: NIMPLY1, lane 4: NIMPLY2, lane 5: XOR1, lane 6: XOR2, lane 7: IMPLY1, lane 8: IMPLY2.
Reference:
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