Team:OUC-China/Engineering
Engineering Design Cycle
Inspiration → Research → Imagine → Design → Test → Improve → Build → Test → Improve → Build → Simulate → Build → Test → Simulate → Design → Simulate → Future
Inspiration:
Our school is located in Qingdao, a coastal city with a wet atmosphere. That’s not good news for computers, for the high air humidity can impact their service life. Our computer equipment often breaks down because of the high humidity. Apart from this, there are also other environmental factors that interrupt its normal function, like, temperature and dust, etc. What’s more, the thickness of silicon chips has long been a factor affecting computers’ performance. In the future, perhaps we need to seek other kinds of computers that can overcome these disadvantages, and a biocomputer is an option.
Research：
The ultimate goal of combining molecular biology and engineering is to realize the biocomputer. Since the electronic computer is so well-developed according to our research, it seemed unlikely that the biocomputer element we research into will easily surpass the calculation speed of electronic computers.
Therefore, biocomputer should demonstrate excellence in another area, such as, high humidity tolerance, high biocompatibility and mass storage capacity. Biocomputer should have special functions which emphasis on biological characteristic and would be complementary with electronic computers eventually. The expertise we obtained has inspired us to consider realizing biological functions with our biocomputer designs. Finally, in the discussion with other iGEM teams in meet up, we settled that our logic gate designs can be applied to detecting viruses.
Imagine：
Our project is not trying to solve a real world problem with practical applications right away, we built elements of a biological computer. Logic gates are the foundation of computing. To build a biocomputer, we need to start from the basic parts built by biological elements. We hope it will have scientific values and can serve as a comprementary role to electronic computers in the future.
Design：
We chose toehold as the ON-switch and three-way Junction (3WJ) repressor as the OFF-switch. And we used it in the construction of logic gates and the design of some more complex circuits.
Test：
We conducted a series of tests on promoters, the ON RNA switch and the OFF RNA switch. The results of these basic tests provided references for the construction of logic gates and the design of some more complex circuits.
Improve：
We hope to improve the stability of the trigger from two aspects to reduce false negatives.
① Add the hairpin at the 5' end of the trigger.
② Knockout/knockdown of RNAase gene (using E. coli BL21 Star (DE3) as biological chassis).
Build：
Based on toehold, 3WJ repressor and existing logic gates, we designed new logic operations (IMPLY gate and XOR gate).
Test：
In the verification of the logic circuit, the inductors corresponding to the promoter before the switch structure were taken as the inputs, and the expression quantity of fluorescent protein was taken as the output, so as to test the existing logic operations and the logic operations designed by us (IMPLY gate and XOR gate).
Improve：
We have developed a SOFTWARE that can calculate the common threshold. Threshold calculation can give the normalized parameters of fluorescence intensity and the calculated threshold, so that the designers can get the common threshold of different logic gates easily and combine different types of logic gates better.
Build：
We used σ factors to implement cascades of different RNA logic gates. We also designed more complex multiple-input logic gates and swapping gates to show more possibilities of logic gates.
Simulate：
We use universal thresholds, promoter data, basic logic gate data and σ factor data to build cascade loops, multiple-input gates and swapping gates.
Build：
We construct the adder and the subtractor with our logic gates. The calculation can be done by adjusting the concentration of trigger RNA. In the future, they can be used in the more complicated calculations.
Test：
Test the logic gates needed (XOR gate, AND gate and NIMPLY gate) to build half adder and subtracter.
Simulate：
We simulate the operations of semi-adders and subtracters with available data.
Design：
Our logic gates also can be used to detect various viruses. We design the logic operations according to the virus sequence. The use of them can reduce false negatives and false positive of virus tests and also makes it possible to detect multiple viruses simultaneously.
Simulate：
We tested the matching degree of the logic operation and the virus sequence by modeling method. The logic operations can identify the sequence well and detect it.
Future：
Under the influence of COVID-19, our experimental time was more limited and the control of virus usage became stricter. Therefore, we didn’t conduct experimental verification on the application of virus detection, but only demonstrated the application of virus detection with the logic gates in the biological computer through design and modeling simulation. We believe that these logic gates have great potential in resistance gene detection, virus detection, and disease detection.
New Parts
We designed and built some new parts, and they worked as expected.
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The XOR is inspired by the NIMPLY gate, consisting of a toehold switch and two triggers.
Figure 1. Demonstration of XOR gate
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. 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.
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We combined the 3WJ switch and toehold switch to realize the IMPLY Boolean calculation.
Figure 3. Demonstration of IMPLY gate
When no trigger is expressed, this logic gate just likes a 3WJ switch. When trigger A expressed, the trigger will bind to the switch RNA. The binding allows for a branch migration process, exposing AUG and RBS for translation initiation. When trigger B expressed, the trigger will bind to 3WJ switch RNA. The resulting trigger–switch complex has a stable 3WJ structure that effectively sequesters the RBS and the start codon within the loop and stem of the switch RNA, respectively, and strongly represses translation. When both trigger A and B are expressed, the binding of trigger RNA to the toehold sequence allows the RNA polymerase to bind to the former RBS and break open 3WJ stable hairpin.
Figure 4. Two-input toehold and 3WJ repressor IMPLY gate
The left figure shows that when INPUT A=0, INPUT B=1, 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.
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We improved the NIMPLY gate in the paper and built new parts.
Figure 5. Demonstration 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 6. 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.