Engineering/Build
Build
On this page you will find information on: Introduction, Wet-lab considerations, In silico considerations, Resources.
Introduction
After following the guidelines in the design stage of the DBTL cycle, you should now have a good idea of what your system is and what you need to build. For wet lab synthetic biology projects, the build stage involves assembling DNA constructs. There are many methods for DNA assembly, and the most appropriate will be influenced by a number of factors, such as resources available and your design specifications. For dry lab synthetic biology projects, the build stage may include 3D-printing designs, soldering electronics, or building computation models/software tools.
Questions to Consider for wet lab projects
Below are some questions which will help guide you in determining your build strategy. A lot of these tie back into your design specification and other decisions made during the design stage.
- Are you building the entire system as a whole, or separate components of the system?
- If you plan to build the system in separate parts for individual testing, will they eventually need to be assembled together? If so, does your build strategy allow for this?
- Will you build all components simultaneously?
- Does your chosen assembly method create scar sites?
- If so, will this impact your design specification and/or downstream function?
- Will you need to generate different variants of your build?
- If you plan on building different variants, you can future proof by building the different parts separately and using a modular cloning standard such as Phytobricks and Loop to assemble them together
- How could you modify your build in future iterations of the DBTL cycle?
- Can you re-assemble from the separate parts? Will you re-synthesise to incorporate any modifications?
- Are you using an appropriate chassis/plasmid for the building stage?
- Could anything in your build be toxic to the chassis? How could you limit this impact?
- Does your build need to be moved to a different chassis/plasmid for testing? How will you accomplish this?
- How can you screen for correct builds?
- Will transformed colonies look different?
- Can you use agarose gel electrophoresis and enzymatic digests to screen for correct digest profiles?
- How will you verify that the builds are correct?
- Will you sequence your build? Do you have suitable primers for this?
- Do you have access to the DNA parts and reagents required for your build strategy?
- How can you screen for correct builds?
- If applicable, will you use automation to help build your parts? If so, what equipment do you need? Do you have access to this equipment?
Once you’ve used these guidelines to determine your build strategy, ensure that your final build still matches your design specification.
After you have built your constructs and verified they are correct, you can then move on to the test stage.
Tips to Get Started
Before ordering any parts or starting to build anything in the lab, ensure that you have a build specification based on the questions above. This will help ensure that what you build is what you need, and allow you to have a good idea of what to do once you are in the lab. This can be aided using in silico design tools such as Benchling. It will also ensure that you know how to screen whether your build was successful or not, and hopefully give you options if it fails.
If your build does fail, go back to the design and ensure that nothing has been overlooked. Do you have the correct restriction sites? Do you have any illegal restrictions sites? Are you building something which might be toxic to the cell? Have you used the correct backbone to assemble into?
Another huge resource to teams is other iGEM teams! Collaboration is an essential part of science and your fellow iGEMers may be able to help you with experimental design, implementation, access to expertise and so much more!
In silico Build Stage
In silico projects and synthetic biology models also have a build stage. This is the section where you begin implementing your in silico tool/software/model. Based on the
Once you’ve used these guidelines to determine your build strategy, ensure that your final build still matches your design specification.
After you have built your constructs and verified they are correct, you can then move on to the test stage.
Tips to Get Started
Starting to implement your model, software tool, or other in silico project can be daunting and knowing where to start can be tricky. Based on your design specification from the design stage, you should have an idea of what your model will look like. Use this as your guide.
When first starting to implement your model, you can be very abstract. For example, you can ignore any parameters or values and just use random placeholders. These can be added in later. You can also start by picking a relatively simple part of the model for example, expression of a protein) and just implementing that at first. Once you’ve managed to model and simulate that small section with placeholder values, you can start to expand the model and add in the other processes around it. You can also start to add in experimentally derived parameters if you have them. Remember that for the first iteration of your model, you might be stuck with using best guesses for some parameters. You might need to use results from your experiments to get the real parameters. This is one way in which your model can inform your experimental design, which is discussed in the next test stage!
Build Stage Resources
Our 2021 and 2020 iGEM webinar series on the following topics may be useful resources for the build stage:
- DNA Assembly Techniques
- Gibson Assembly and Yeast
- Transformation and Sequencing
For 2021, IDT is once again providing iGEM teams with gBlocks to help with building your constructs. They have a number of resources on their iGEM page, and also a useful webinars on how to use gBlocks:
There are also several tools which can help you with the Build stage:
- Benchling: An online tool to help you create, edit, store, and share DNA and protein designs. It also includes tools to simulate molecular biology procedures such as restriction digest, PCR, and DNA assembly.
- NEBuilder: An online tool which can be used to verify if your gibson assembly is likely to work, and can also help you design primers for gibson assembly if needed
- Virtual PCR: An online tool for learning how to perform PCR
- Protocols.io: A repository of open source protocols with tips and user comments. Can be used to find alternative protocols for DNA assembly or other procedures required for the Build stage.
- Matlab: A programming platform which can be used to model various aspects of your project (from biological systems to hardware). Available for free to iGEM teams. Also provides tutorials and webinars to learn how to use Matlab in a synthetic biology context.
- SBML: A language for coding biological models.
- Antimony: A Python package for modelling biological systems using SBML shorthand.
- COPASI: A software tool for mathematical modelling. Can be used to create and simulate models. Also has the option to import SBML models.
- GRO: A programming language for modelling communities of cells. Tutorials and teaching resources are available.
Once you’ve used these guidelines to determine your build strategy, ensure that your final build still matches your design specification.
After you have built your constructs and verified they are correct, you can then move on to the test stage.
Please email the Engineering Committee at engineering [AT] igem [DOT] org and provide links to material with a short description. We’ll check it out and if we believe it will be helpful, we’ll add it to this page!
iGEM Foundation
The International Genetically Engineered Machine (iGEM) Foundation is an independent, non-profit organization dedicated to the advancement of synthetic biology, education and competition, and the development of an open community and collaboration. Visit us at igem.org
Contact
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