Team:OUC-China/Implementation

• PROJECT
  • Description
  • Design
  • Results
  • Contribution
  • Engineering
  • Implementation
  • Proof of Concept
• WET LAB
  • Experiments
  • Safety
  • Measurement
• MODEL
  • Overview
  • 3WJ
  • ODE
  • Sequence Design
  • Software
• PARTS
  • Overview
  • Improvement
  • Collection
• HUMAN PRACTICES
  • Human Practices
  • Education
  • Comics
• COLLABORATIONS
  • Collaborations
  • Meet Up
  • Team List
  • Partnership
• TEAM
  • Members
  • Attributions

Overview

Our project is not trying to solve a real world problem with practical applications right away, we built elements of a biocomputer. Logic gates are the basis 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 complementary role to electronic computers. As for how to implement our projects in the real world, we hope to reflect on the application value of our project from the two aspects of calculation and detection.

Usage

Based on the RNA switch, we constructed different logic gates. We use the trigger as the input signal, protein as the output signal, and switch as the intermediate processing element. The logic operations we designed can be applied to different systems. Our switch can be connected behind different types of promoters to give them logical functions. Our logic operations can also combine with other intermediate substances to achieve cascading. Our logic gates can handle not only two inputs, but also complex multiple inputs. In addition, they have a richer form of swapping gates. These can be flexibly applied for different purposes. If you want more details, see the DESIGN and RESULTS.

We also hope that the application of calculation can give those scientists who are devoted to biocomputers new ideas and motivate them to generate unique designs. And due to these parts can work in a wet environment, in a few years, we may be able to implant a biocomputer chip in the human brain.

Safety Aspects

We are convinced that we are responsible for the environment and our colleagues in the experiment. All the parts we have utilized are selected from Risk Group 1 in our design. We worked with E. coli DH5α, E. coli BL21 (DE3) and E. coli BL21 star (DE3), which are both non-pathogenic bacteria. All strains are on the Whitelist and were provided by the company. For the sake of environments, all the chassis organisms will be sterilized before being abandoned to prevent the genes from leaking.

As a matter of fact, the RNA-based regulatory elements and the logic gates system we built are harmless for the growth of bacteria and relatively safe for applications.

Challenges

There are many kinds of logic gate systems, which are built by various components, such as DNA、CRISPR-dCas9、repressor protein and so on. Despite these developments, one of the potential problems in building a biological computer remains the composability of genetic circuits, the limited number of high-performance components, and the difficulty of integrating multiple components into a large, complex synthetic network.

RNA-based regulatory elements offer a potential solution to this component bottleneck. In the first place, RNA corresponds to a number of potential regulatory elements, and it shows an unprecedented degree of orthogonality. 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. What’s more, mRNA can translate protein outputs directly without transcription, which greatly saves time.

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

①For the optimized 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.

②For the construction of logic gates, we collected previous logic gates that consist of toehold switch. And using toehold switch and 3WJ repressor design the types of double-input logic gates that are still lacking.

③For non-uniform representation of logic gate measurement standard, we made a small program to the standardized fluorescent output of logic gates.

④For showing more complex functionality, 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.

In the future, we may need to apply it in a cell-free system to reduce the limit of metabolic pressure, which will present a number of new challenges. First, there are some difficulties in the preparation and storage of cell-free systems. We may need to find an easier way for long-term preservation and a cheaper method to realize it. Second, RNA is unstable in vitro and degrades easily at room temperature. So, we also need to find a method that can maintain the normal function of RNA to ensure the smooth implementation of our logic gate.

Promising Applications

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. 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. Logic gates, the building blocks of biological computers, have been shown to be useful for resistance gene detection, virus detection and disease detection. Among them, we have presented the design related to virus detection in the DESIGN. We believe they still have great potential and they can show their value in a much broader field.