Team:ICT-Mumbai/Software
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BioBrick parts and standard assembly
A BioBrick part can be defined as a DNA sequence (encoding a particular biological function) that conforms to a set of standard rules. The Registry of Standard Biological Parts consists of over 2000 standard parts, each of which can be assembled according to BioBrick assembly standards to create multi-component parts of increasing complexity and functionality (Shetty et al., 2008).
There are different types of assembly standards, namely BioBrick Standard Assembly (RFC 10), BioBrick BB-2 Assembly (RFC 21), Silver/Fusion Bricks (RFC 23), Freiburg Fusion Protein (RFC 25) and BioBrick MoClo (RFC 1000). (Registry of Standard Biological Parts, iGEM; Knight, 2007; Knight, 2008; Anderson et al., 2009; Philips et al., 2006; Grünberg et al., 2009).
The Biobrick assembly standard RFC 10 is the most widely used assembly standard (Registry of Standard Biological Parts, iGEM). Each year hundreds of iGEM teams all over the world contribute and submit BioBrick compatible parts to the registry. The BioBrick standard has a set of stringent requirements for a part to be RFC 10 compatible:
RFC 10 standards
| Sequence | Type | Enzyme |
|---|---|---|
| gaattc | Illegal | EcoRI |
| tctaga | Illegal | XbaI |
| actagt | Illegal | SpeI | ctgcag | Illegal | PstI | gcggccgc | Avoid | NotI |
- The part must not contain any of the following illegal sites:
- EcoRI
- XbaI
- SpeI
- PstI
- The part must not contain the following avoidable site:
- NotI
- The part should have a Biobrick prefix for
- Non-coding parts
- Coding parts
- Modifications for parts having start codon other than ATG
- Terminator sequence for coding parts
- The part should have a Biobrick suffix.
To ensure that the biological part conforms to these standards, researchers have to carry out multiple checks and modifications, some of which include:
- Checking for presence of illegal and avoidable restriction sites in the part.
- Modification of part sequence to replace illegal and avoidable restriction sites, depending on chassis organism.
- Addition of suitable prefix according to standards described above.
- Addition of suitable suffix according to standards described above.
BBrickIt: The one-stop solution to your BioBricking needs
Based on our own experience and a survey that we conducted with all iGEM 2018 teams (results can be found here) we found that one has to alternate between different programs to achieve all requirements to construct a BioBrick.
We found ourselves alternating between different programs such NEB cutter, various codon optimization tools and the iGEM registry. Although this process is repetitive and tedious (more so when creating/submitting many parts), it also is very important for the sequence that is generated to be free of errors, the chances of which increases when done manually.
We sought to create an open source on-line tool which would enable users to check, modify and validate their parts according to RFC 10 standards, all in one single program thereby making the entire process simpler, faster and error free. We believe that this tool will be particularly helpful to first-time participants of iGEM.
BioBrick your part and more!
Identify and validate part for presence/absence of illegal restriction sites
Replace sequence according to chassis (E. coli, B. subtilis and S. cerevisiae)
Add required Prefix and suffix
Available in Mac and Windows versions
Key considerations while designing the software
- Open source
- Ease of use
- Single program for all analysis and design
- Error-free sequence modification
- Compatible for common chassis organism
User guide
We have created a user guide that outlines step-by-step procedure for using this software. The user guide can be accessed here.
BBrickIt Software Beta Testing
Several iGEM teams tested our software along the way.
Many teams asked us to make a comprehensive user manual for this software and without any delay we created one and shared with all of them! Also, initially, we shared an .exe file on Github, which worked only on Windows operating system. Many teams use MacOS as their major operating system. Taking their suggestions into account, we immediately started working on Mac version of the software. Now MacOS version of BBrickIt is also available in GitHub repository.
Target audience: The iGEM community!
Feedback from iGEM teams
Numerous teams tried out our software and we got superb responses from many of them.
Kalyani from iGEM UCSD said commended our software saying, “Amazing Work!”
iGEM IISER Kolkata were extremely happy to “bypass the hectic job of having to think of prefix and suffix sites, scars, illegal sites etc. before planning to clone anything in the standard vectors!” They used our software with 5 genes that were part of their project, and it worked amazingly well & without any bug.
Kyle from iGEM Washington University, St. Louis said, “The tool is easy to use and well outlined.” This software would have helped them catch that they initially forgot to add the BioBrick prefix and suffix to their gene sequence!
iGEM ECUST said, “BBrickIt is a useful tool for iGEM teams in producing complete BioBricked part and is also very efficient.”
Methodology
The initial idea for this software came to us while we were shuttling between various softwares in order to identify the compatibility of our sequence as per BioBrick RFC 10 Standards. We shuffled between various tools for performing tasks like identifying illegal restriction sites, taking a reverse complement of the sequence, and manually adding prefix and suffix. We then decided to create a simple, user friendly GUI that can perform all these functions and specifically look for sites as per BioBricking standards.
After the initial version was made, we decided to add to it the amazing feature of suggesting a replacement sequence in case your sequence is not compatible for BioBricking. We also decided to add to it the functionality of suggesting sequences that are optimized for the organism of origin. Currently the program works for three commonly used chassis organisms namely E. coli, B. subtilis and S. Cerevisiae.
This program was coded purely using Python language and the GUI created via the tKinter library that is provided along with it. This is an open source program and its source code can be found here.
References
Software developed by: iGEM Team ICT Mumbai 2018.