Inspiration

Computers have changed a lot in the past a hundred years, initially, they were only for researches, gradually they became available to families, now we can even see people carrying tablet computers walking in the street. Despite the maturity of the computer industry and computers’ well-acceptance in the public, there are still some disadvantages lying in its structure.

For example, Qingdao is a coastal city with a wet atmosphere. And that’s not good news for computers, for the high air humidity can impact their service life. Apart from this, there are also other environmental factors 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 biocomputer is an option. The CPU of electronic computers is based on the connection of logic gates, so many scientists believe that if the same logic gates can be constructed with biological components, it’s likely that the structure will function as a computer.

So, in the year 2020, OUC-China is inspired by biocomputer. Our project aims at building logic gates with two kinds of RNA switches, and apply them to calculation and detecting viruses.

For more details of our project click on Design

Research Background

The Synthesis of a variety of biological circuits for specific functional purposes has made tremendous progress in recent years. The ultimate goal of combining molecular biology and engineering is to realize the biocomputer ^[1]. In the traditional electronic circuits, one uses several Boolean logic gates to realize a CPU. Before constructing a biocomputer in the genetic system, we started here by constructing a class of genetic logic gates ^[1].

There are many kinds of logic gate systems, which are built by various components, such as DNA^[2-4]、CRISPR-dCas9 ^[3]、repressor protein ^[1] and so on. Despite these developments, an underlying problem in constructing a biocomputer, which is the same as the problem in synthetic biology, remains 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 ^[5].

The Useful Application of Toehold Switch and 3WJ Repressor

RNA-based regulatory elements offer a potential solution to this component bottleneck. In the first place, RNA corresponds to many potential regulatory elements. Thus, the sheer diversity of possible RNA-based parts suggest that previous devices have not come close to realizing the potential of highly orthogonal regulation. In the second place, Biological parts constructed from RNA take advantage of predictable Watson-Crick base pairing to control cellular behavior and can harness sophisticated software tools for predicting RNA-RNA interactions ^[5]. What’s more, mRNA can translate protein outputs directly without transcription, which greatly saves time.

Common RNA elements that can implement ON/OFF are riboregulators、toehold switch and so on. A common limitation for riboregulators has been their dynamic range. Previous prokaryotic translational riboregulators have typically modulated biological signals by up to a maximum of ~55-fold for activators and up to ~10-fold for repressors ^[5]. In contrast, protein-based transcriptional regulators have demonstrated dynamic ranges over an order of magnitude higher, with widely-used inducible promoters regulating protein expression over 350-fold and sigma factor-promoter pairs providing up to 480-fold modulation ^[5]. However, the protein-based transcriptional regulators have a problem with high metabolic pressure. Toehold switch and 3WJ repressor offer a potential solution to these problems. Typical toehold switch and 3WJ repressor employ interaction domains consisting of ~100 nt, which has low metabolic pressure. And the toehold switch exhibits the ON/OFF ratio exceeding 400, which can meet a large dynamic range.

Our Research

By reviewing the existing problems and solutions towards them, we are aware of some aspects are worth being researched and optimized.

For the optimize the dynamic range of the switch, we designed the 5 '-end hairpin to improve the stability of RNA. The 5’-end hairpin can block the RNase so that the RNA will be accumulated and the ON/OFF ratio will be raised.

For the construction of logic gates, we the collected the previous logic gates that consist of a toehold switch or 3WJ repressor. And using both toehold switch and 3WJ repressor design the types of double-input logic gates that are still lacking. We had also attempted to construct logic gates with multiple inputs and cascading them using the σ factors. We also tried to design the swapping gates which can switch between two kinds of logic gates.

For the non-uniform representation of logic gate measurement standard, we made a small program to the standardized fluorescent output of logic gates.

Summary

This year, OUC-China made the project named “LOGITCH”, which hoped to provide basic components and debugging parameters with excellent performance for biocomputer and to lay a foundation for the development of biocomputer in the future.

We used two types of switch: toehold (on-switch) and 3WJ repressor (off-switch) which are both highly regulated at the post-transcriptional level. We integrated the logic gates that consist of toehold and 3WJ repressor. And we designed and tested the currently lacking types of double input logic gates. In order to standardize the fluorescent output of our logic gates, we make a small program to calculate the coefficients that should be multiplied by different logic gates’ output.

We also designed swapping gates that can switch between two kinds of logic gates and tried to construct logic gates with multiple inputs and cascading them using the σ factors. By doing these, we want to enrich types of logic gates and implement complex calculations.

Finally, 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.

COVID-19 Impacted On Our Project

In the spring semester, our school was closed because of the COVID-19. And because our team members are scattered across the country, we have to conduct our meetings online. Online meetings are not conducive to communication, so we tried a lot of methods to improve our efficiency.

Our experimental plans also failed to implement as intended. Because we don’t know when can we go back to school, so we made many experimental plans which star at a different time and kept cutting out experiments that aren't particularly important.

Fortunately, some team members of us got back to school on July 23rd. Our experimental work was carried out.

References

[1]Chun-Liang L , Ting-Yu K , Wei-Xian L . Synthesis of control unit for future biocomputer[J]. Journal of Biological Engineering, 2018, 12(1):14-.

[2]Siuti, P., Yazbek, J. & Lu, T. Synthetic circuits integrating logic and memory in living cells. Nat Biotechnol 31, 448–452 (2013). https://doi.org/10.1038/nbt.2510

[3]Gander, M.W., Vrana, J.D., Voje, W.E., Carothers, J.M., Klavins, E., 2017. Digital logic circuits in yeast with CRISPR-dCas9 NOR gates. Nat. Commun. 8, 15459.

[4]Qian, L., Winfree, E. & Bruck, J. Neural network computation with DNA strand displacement cascades.Nature 475, 368–372 (2011). https://doi.org/10.1038/nature10262

[5]Green et al., Toehold Switches: De-Novo-Designed Regulators of Gene Expression, Cell (2014), http://dx.doi.org/10.1016/j.cell.2014.10.002