Development of Genetic Design Automation Tool to Program the Living Cell

Project Title

Development of Genetic Design Automation Tool to Program the Living Cell

Project Area of Specialization NeuroTechProject Summary

Genetic circuits are an application of synthetic biology where biological parts inside a cell are designed to perform logical computations like digital electronic circuits. These circuits are composed of genetic components found in DNA and operate inside living cells, which execute desired logical operations activated by the presence or absence of certain proteins or other species. They are represented in the form of SBOL (Synthetic Biology Open Language), a standardized format to represent the structural and functional aspects of biological systems.

How genetic circuits are different from electronic circuits?

• Controls the behavior of a living cell
• Composed of a genetic component of DNA (e.g. promoter)
• The behavior of genetic circuits is defined in base pairs (e.g. ATGC...) opposite to the electronic circuits defined in bitstreams (e.g. 1010...)
• Triggered by external signals such as light, chemicals, proteins, etc. to produce the output protein
• Intermediate signals are molecules which exhibit high cross-talk with the neighboring components
• The input and output quantities are different as compared to electronic circuits (volts)

These circuits are generated in Wet Lab which is very time consuming and in case of any mishandling of the equipment or any kind of errors, the experiment needs to be repeated all over again. It not only consumes so much time, but it can be very costly in some cases if the experiment is being conducted with many different components and at a high level. Many such tools have been developed to generate, synthesize and simulate genetic circuits. The existing tools are limited to the users who have the users who have background knowledge about the SBOL and they need to analyze the SBOL model to understand the circuit. However, learning SBOL is quite complicated. Moreover, if the tool is being used by a biologist in order to verify the circuit, they should not face any difficulty in order to get the output circuit.

The aim of this project is to provide the user a very high level of abstraction by making the tool very user friendly and simple to operate in such a way that user does not need to know the depth and details of the components, concepts related to both synthetic biology and engineering, or of any kind of programming, if required any. It is being programmed with the motivation that it should be as easy as a user who just knows a little concept of logic gates can develop the genetic circuits easily.

Project Objectives

Nature is using genetic logic circuits to regulate the fundamental processes of life. Due to stochastic nature, there is a growing interest in being able to analyze and verify the logical behavior of a genetic circuit model, prior to its physical implementation in a laboratory. The main objective of the project is to develop a Genetic Design Automation Tool, which can analyze that behavior.

This work is originally inspired by the processes of optimization and technology mapping of electronic circuits in the electronic design automation (EDA) industry. In electronic design automation for digital electronics, the combinatorial circuit optimization is always required to implement the circuit with the minimum number of logic gates. This area-efficient implementation of digital circuits not only helps to reduce the size of electronic devices but also avoid wasting power and redundant resources.

The tool will be able to generate all the possible circuits of the entered Boolean expression, with respect to the provided constraints. The circuits will be generated by going through a certain number of steps/methodology, which is discussed in the implementation section. The constraints include total time taken to propagate the circuit and the maximum number of gates in the generated circuits. These constraints are crucial because implementing a larger circuit would increase the metabolic burden inside the cell as it would require more cellular energy to maintain its presence inside the cell and increase the time taken to process it which means decreasing the efficiency as well. Moreover, if the limit is not defined, the tool will generate all the possible circuits and the user will have to scrutinize all the circuits manually and select the best suitable circuit. Hence, for a larger set of circuits, this is time-consuming and impractical. At the end of this project, this updated tool will be available for public usage. For the inclusion of the parameters of the threshold value and propagation delay, we will be going to CECOS Lab in Peshawar during the summers. We will be generating circuits in the wet lab and gaining the knowledge of the physical implementation of the circuits. Those parameters will be updated in the libraries and the algorithm will be developed accordingly. Moreover, the tool will be able to generate the SBOL Data file, which is the standardized form to represent the structural and functional aspects of biological systems, that could be used in any other software to verify the simulating behavior of the genetic circuit.

Project Implementation Method

The tool will be able to process the input Boolean expression in the form of Sum of Products (SOP) or Product of Sums (POS). If the entered expression is in the form of POS than it will be converted to SOP and then the expression will be first optimized followed by the synthesis and technology mapping. The simplification of the expression is required so that there are fewer gates to deal with in the final obtaining circuits. In the view of genetic circuits, it also makes sense to have less number of gates in the circuits because, the larger the circuit, the more metabolic burden it is as it would require more cellular energy to just stay inside the cell, which would result in decreasing the efficiency of the circuit itself and the time taken to process it. The gates in the reduced circuit will then be converted to NOT/NOR form because these are the basic gates and the genetic gate library available only contains NOR and NOT gates. Digital logic gates are very easy to connect with each other because those gates have the same physical quantity i.e. voltage as their input and output but genetic circuits have different input and different output. The algorithm, technology mapping, will checks if a gate is compatible to be connected with the other gates are not i.e. the output of a gate is compatible with the input of the following gate.

The above-mentioned library was developed by Cello, which doesn’t contain any kind of parameters regarding the threshold value, categorizes the analog concentration levels to digital logic levels 0 and 1, and the time required to generate a specific output. We will be visiting CECOS lab to generate circuits in the wet lab and the measured parameters will be added in the library and the algorithm will be developed accordingly. The importance of these parameters has been described in the previous section of objectives.

The other main objective will be the visual representation of the genetic circuits using SBOL. That will help the user understand and compare the SBOL notation with the electrical logic representation.

Benefits of the Project

The tool will provide the high-level abstraction for users to generate genetic circuits without any backhand knowledge of SBOL, just by entering the Boolean expression as an input for which, a basic knowledge of logic gates is required to write the Boolean expression. This would be a common platform for biologists, to generate the Genetic circuits, and for the people who want to pursue a field related to Bio-medical sciences, where they can learn the basics of genetic circuits by generating them and analyzing them. This tool would also be used to educate students about the field of genetic circuits and the way to represent them in the form of SBOL, as the output will also be in the form of SBOL visual representation. The output data file, the SBOL Data file, could be used in any other software, such as DVASim, to analyze the behavior of the genetic circuits thorough real behavior simulation. The real behavior parameters will be generated from the updated library of our tool.

Technical Details of Final Deliverable

1. Optimization algorithm: An algorithm to simplify the expression using a meta-heuristic approach.
2. Synthesis algorithm: To convert the simplified expression to NOR/NOT form.
3. Technology mapping: The algorithm to generate the circuits by checking the compatibility of the gates from the library.
4. Representation: The output will be represented in the following three different ways.
   • SBOL visual representation: A SBOL visual circuit will be generated for every given input.
   • SBOL Data file representation: A SBOL data file will also be generated.
   • Electronic circuit representation: An equivalent electronic circuit will also be generated
5. User Interface: 
   • Interactive GUI
   • Script (command) support: The tool will also support the input in the form of a script (command).

6. Additional Libraries:

   • Wet lab development of genetic AND/OR Gate: These libraries will be created with the help of tested circuits in the Biological Lab in Peshawar CECOS, in collaboration with Dr. Faisal.
   • Measurement of the threshold value, propagation delay, degradation rate values.
   • Integration of real parameters in the tool: These real parameters will be obtained with the collaboration of CECOS and they will be integrated with the library.

7. Simulation/Verification:

   • Functional verification of circuits in DVASim.
   • Generating SBML file (tentative):  In order to verify a circuit, DVASim requires the input file in SBML format. For the format conversion, iBioSim is used. Instead of converting the file format using iBioSim, we will generate the SBML file format directly from our tool and then use it for verification in DVASim.

8. Public Usage: At completion, the tool will be made available for free public usage. For that, we need to purchase a web domain.

9. Software User/Developer's Guide: Along with the tool, at the completion, a software developers’ guide will be released for future developers and users.

10. Additional Input form: An additional truth-table based input option will be generated. User will have an option of inserting the values in the truth table as an input instead of writing an input statement/expression.

11. Research Publication: Manuscript would be ready/submitted at the end in a conference/journal/workshop.

Final Deliverable of the Project Software SystemType of Industry IT , Medical Technologies NeuroTech, OthersSustainable Development Goals Good Health and Well-Being for People, Quality EducationRequired Resources

Item Name	Type	No. of Units	Per Unit Cost (in Rs)	Total (in Rs)
			Total in (Rs)	80000
DNA and Polymerases and PCR	Equipment	2	10000	20000
Web Domain Purchase (actual price Rs. 13000)	Miscellaneous	1	10000	10000
Restriction endonucleases	Equipment	2	12500	25000
DNA Ligase enzymes	Equipment	2	12500	25000