Team:OUC-China/Poster

Poster: OUC-China

Logitch: Logic Gates and RNA Switch

Presented by Team OUC-China 2020

Chaoxin Chen¹, Qi Wu¹, Qingying Ke¹, Yifan Zhang¹, Ting Wang¹, Feiwen Wei¹, Yue Niu¹, Chenguang Liu², Xiaolong Wang², Guanglei Liu²

¹iGEM Student Team Member, ²iGEM Team Primary PI, Ocean University of China, Qingdao, China

Abstract 

In the future, more work must be done by computer, such as detecting viruses and monitoring health in vivo. Such a function can only be achieved by biological computers working in a wet environment, which plays an important role that traditional computers cannot replace. Since the basic functional unit of an electronic computer is implemented through logic gates, we incorporated a series of logic gates which were built by toehold and 3WJ repressor and supplemented current lack of types with our design. We also built more complex multi-input logic gates using basic logic gates. In order to show the wide application prospect of these logic gates, we used them in the construction of adder and subtractor, as well as virus detection. Biocomputer may have various possibilities in the next few years, and we hope our project can have some impact on its development.

Description

Computers have changed a lot in the past a hundred years and became an important part of our lives. However, there are still many environmental factors that interrupt its normal function, such as the wet atmosphere, temperature, and dust. So scientists are devoted to seeking other kinds of computers that can overcome these disadvantages, and a biocomputer is an option.

In the traditional electronic circuits, one uses several Boolean logic gates to realize a CPU. There are many kinds of logic gate systems, which are built by various components, such as DNA, CRISPR-dCas9, repressor protein and so on. However, the limited number of composable, high-performance parts for constructing genetic circuits and difficulties that arise when integrating multiple components into a large, complex synthetic network still hinder its development.

Objectives

This season, we aim to…

Collect and complement types of logic gates

Optimize the dynamic range of RNA switch

Achieve composability and multiple-input

Make contributions to the future development of biological computers

Design

In order to achieve our goal, we integrated and designed a series of RNA logic gates. Then, we improved them and finally made them work in practical applications.

By searching the literature， we collected OR, AND, NOT, NOR and NAND gate. And we design IMPLY and XOR gate to fill the existing gap.

Improvements

① Stability

We add the 5’ end hairpin to the trigger RNA blocking the degradation mediated by RNase E to optimize the ON/OFF ratio of the toehold switch.

② Versatility

We developed SOFTWARE to normalize our experimental data. So that we can easily find the common threshold of different logic gates and better combine different types of logic gates.

③ Composability

We will use σ factors to achieve composability. The σ factors have several highly orthogonal pairs so that we can realize sophisticated layered circuits.

④ Multiple-input

We also designed the three-input logic gates and four-input logic gates on the basis of single-input logic gates and two-input logic gates. These enrich the types of our logic gates.

⑤ Swapping Gates

We thought the swapping gate is an interesting and novel idea, so we designed two ways to achieve that. In the first one, we used a trigger to control the transformation between two logic gates. In the second one, we used different triggers to control the transformation.

Result

We designed IMPLY and XOR gate to fill in the existing gap. 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 logic operations (IMPLY and XOR) designed by us.

① IMPLY Gate

We combined the 3WJ switch and toehold switch to realize the IMPLY gate. When only trigger B is expressed, the switch is off. In other cases, the downstream gene can be expressed. The data we measured corresponds to the situation described in the truth table.

② XOR Gate

The XOR gate consists of a toehold switch and two triggers. When input one of these triggers, the switch can turn on. When input these two triggers simultaneously, the switch will still be off. The data we measured corresponds to the situation described in the truth table.

In addition, we add the hairpin structure at the 5' end of triggers to improve the trigger stability, design the software for calculating the threshold to improve the universality and use trans-action factors such as σ factor to improve the compatibility. We also designed more complex multiple-input logic gates and swapping gates to show more possibilities of logic gates. The results of these sections will be presented in the Model.

Model

We developed a score function to rank 3WJ repressors by potential GFP fold reduction. Through calculating by using the MATLAB, the function displays the best parameters linear regression, which provides a coefficient of determination R² of 0.6393.

We established the ODE model to simulate the whole process of RNA switch based on the experiment data. The modeling results are of the same order of magnitude as the experimental results. And according to sensitivities analysis, we found the degradation rate plays an important role in the ON/OFF ratio. So we design the 5‘ end hairpin.

Using NUPACK, we designed the sequences that we wanted. Such as the Trigger with 5’ end hairpin, IMPLY, XOR and NIMPLY. They are all verified by experiments. We also designed the swapping gates and multiple-input switches, which perform well analyzed in NUPACK with different input methods.

Software

We designed a small software to standardize the experimental data. By inputting the experimental results of different logic gates, this program can calculate the coefficient of fluorescence amount of each logic gate, and give the fluorescent light threshold of the final judgment logic gate 0/1.

Parts

Improvement：

We added 3WJ to the T7 promoter (BBa_K2150031) to build an OFF-switch. Our new part (BBa_K3328000) has the opposite function of the T7 promoter. In addition, is an integral part of NOT and IMPLY boolean calculation.

Building:

① IMPLY Gate (BBa_K3328041)

We combined the 3WJ switch and toehold switch to realize the IMPLY gate. When only trigger B is expressed, the switch is off. In other cases, the downstream gene can be expressed.

② XOR Gate (BBa_K3328035)

The XOR gate consists of a toehold switch and two triggers. When input one of these triggers, the switch can turn on. When input these two triggers simultaneously, the switch will still be off.

Collection：

We constructed numerous parts that can perform logical functions and collected parts for six types of logic gates (OR, AND, NOT, NIMPLY, IMPLY, XOR). Once the basic components of our design are put together, they can well form logic gates.

Table1: Basic Parts Collected by OUC-China

Table2: Composite Parts Collected by OUC-China

Methodology

Conventional method of measurement

In the verification of the logic circuit, the inducers corresponding to the promoter before the switch structure were taken as the inputs (after adding aTc, trigger A will be expressed and after adding HSL, trigger B will be expressed), and the expression quantity of fluorescent protein measured by the plate reader was taken as the output. We referred to the normalization formula in the literature and used it to eliminate the effect of OD. After processing the data, we can demonstrate whether the existing logic operations and the logic operations designed by us (IMPLY gate and XOR gate) match the truth table as expected.

(Fexptl/Absexptl) − (Fn.c./Absn.c.)

Two extensible methods of measurement

In view of the fact that we had to borrow the plate reader and sometimes the data was out of range of the apparatus. We start from two aspects, hoping to solve these problems.

Firstly, we solved the problem of the lack of a plate reader in the laboratory to measure fluorescence. We used image processing software with UV-light to obtain qualitative and quantitative results by analyzing fluorescence’s luminance.

Secondly, the method of normalized fluorescence and dilution ratio was used to obtain the fluorescence which is out of range of the plate reader. We selected a data with higher value in the original table with overrange data, and took it as the baseline, then got a ratio of other data on the table to the baseline value. On the new table (after dilution) we did the same data processing. Took the fluorescence ratio of the original table as the x axis, and the fluorescence ratio of the new table as the y axis. After linear fitting, the regression coefficient was found close to 1, so the overrange fluorescence value was estimated by the ratio.

Future

Biocomputers should have special functions and would be complementary with electronic computers eventually. We hope to reflect on the application value from the two aspects of calculation and detection. We believe that these applications will become a reality in the future.

Calculation

① Half-adder

We used the AND gate and XOR gate to make up the half-adder. The half adder is able to add two single binary digits and provide two outputs S (sum) and C (carry).

② Subtractor

We used the NIMPLY gate to make up the subtractor. This subtracter can simulate S1-S2=Ps, and Ps is the fluorescence intensity of the reporter gene.

Detection

① Reduce False Negative

Using OR gate can effectively reduce false negative, 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.

② Reduce False Positive

The use of the AND gate containing an inhibiting hairpin increased the specificity of the detection. When both homologous and specific fragments are detected, the reporter gene is expressed.

③ Multi-virus Detection

In complex environments, there is often more than one virus. By designing particular regulatory elements, we could detect multiple viruses simultaneously in an orthogonal way.

Human Practice

We carried on interviews with the professors in our school. We hoped professors would evaluate our project and give us some opinions on how to improve it.

We first sought help from professors in Computer Science and Technology from the College of Information Science and Engineering with e-mails. They offered some opinions about the relationship between biocomputers and electronic computers: maybe our project is not so practical at present, but it does have scientific values and can serve as a complementary role to electronic computers in the future.

Also, the professors gave us some valuable opinions on how to improve our project. Professor Bo Qin suggested us emphasize the stability of our design, therefore, we added a 5’ end hairpin design on the trigger RNA to block degradation. Under professor Haipeng Qu’s suggestion, we tried to design cascades and multiple-input logic gates for a further step.

In order to learn more about the concept and characteristics of biocomputers, we established contact with professor Xiaolong Wang, who used to research biocomputers. Professor Xiaolong Wang’s idea echoed with our previous interviews: biocomputer should have special functions and would be complementary with electronic computers eventually. Considering his advice, we constructed logic gates that can swap between two Boolean logic types and tried to apply our design to virus detection.

Last but not least, by interviewing stakeholders, who are professionals or students in the computer industry, we confirmed the public’s expectation of the development of computer and biocomputer applications on detection.

Education

Questionnaire analysis

We have determined several ways of education suitable for children in poor areas through the investigation.

Comics

We reified and simplified the knowledge of synthetic biology in the form of caricatures to stimulate the learning interest of children in poor areas.

Live broadcasting

We developed an impeccable and sustainable live lecture plan after early communication with teachers and combining with the actual situation of local students.

Videos

We infiltrated biology knowledge in all the aspects of children's life through videos in three aspects, including basic knowledge of biology, synthetic biology and marine biology.

References and Sponsors

References

[1] Green, A. A., Kim, J., Ma, D., Silver, P. A., Collins, J. J., & Yin, P. (2017). Complex cellular logic computation using ribocomputing devices. Nature, 548(7665), 117–121. doi:10.1038/nature23271

[2] Kim, J., Zhou, Y., Carlson, P. D., Teichmann, M., Chaudhary, S., Simmel,F. C., … Green, A. A. (2019). De novo-designed translation-repressing riboregulators for multi-input cellular logic. Nature Chemical Biology. doi:10.1038/s41589-019-0388-1

[3] Green, A. A., Silver, P. A., Collins, J. J., and Yin, P. (2014) toehold switches: de-novo-designed regulators of gene expression. Cell 159, 925– 939, DOI: 10.1016/j.cell.2014.10.002

[4] Meyer, S., Chappell, J., Sankar, S., Chew, R. and Lucks, J.B. (2016), Improving fold activaton of small transcription activating RNAs (STARs) with rational RNA engineering strategies. Biotechnol. Bioeng., 113: 216-225. doi:10.1002/bit.25693

[5] Song, T., Garg, S., Mokhtar, R., Bui, H., & Reif, J. (2016). Analog Computation by DNA Stran Displacement Circuits. ACS Synthetic Biology, 5(8), 898–912. doi:10.1021/acssynbio.6b00144

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