Team:IIT-Madras/IntegratedHP
Integrated Human Practices
Codon bias table generation for A. baylyi:
Background:
During the ideation process for the project for 2018, we decided to study the degradation of lignin to produce vanillin which has higher economic value. For this, we conducted a literature survey regarding the degradation of the aromatic rings of lignin.
We found several organisms capable of degrading such aromatic compounds. Some organisms such as E. coli and L. lactis. are popular synthetic hosts and require transformation. After a brief literature survey, we found several ‘naturally competent’ microorganisms. However, most of them were reported to be pathogenic and are known to harmful for human use. Being a laboratory that conforms to a biosafety level of 1, we could not work with those organisms.
After several discussions and literature surveys, we decided that the best organism that conforms to our biosafety level and is a naturally competent host is Acinetobacter baylyi. The ADP1 strain of this organism is naturally competent, non-pathogenic, and has the property of degrading aromatic compounds. This strain can be purchased from DSMZ. While perusing literature for Acinetobacter baylyi ADP1 we couldn't find substantial material, as information was sparse and not many tools were available to undertake synthetic biology experiments with this organism. This motivated us to build a toolbox for Acinetobacter baylyi that was generic and hence could effectively be used by research groups working with other organisms as well.
Problem:
A primary requirement for experimentation in synthetic biology is a fluorescent reporter protein. Hence we planned to codon-optimize two reporter proteins, GFP and mCherry, for A. baylyi. When we approached companies for codon-optimized reporter proteins they got back to us saying that they did not have a codon bias table available for A. baylyi for our strain.
Our solution:
We made a freely available online tool called CUTE (Codon Usage Table Enumerator). This generates a codon usage table by taking the protein coding annotation into consideration. This tool can be used for all organisms whose genome has been sequenced and whose protein coding regions are annotated. CUTE can be found on the CUTE ChassiDex website. We generated the codon usage table from the protein annotation of A. baylyi available on the NCBI website. Using this codon table, we codon optimized reporter proteins and the characterization can be found here. Following this we conceptualised our next tool - a synthetic promoter library for Acinetobacter baylyi ADP1. We designed a T5 promoter-based library which can exhibit varying expression strengths. Our aim was to make this library as generic as possible so as it maximise its use in synthetic biology.
Results:
From our fluorometry experiments, we can clearly see that Codon optimized GFP shows higher expression than other GFP. This clearly demonstrates that expression of codon optimized GFP is higher than GFP itself. This suggests that codon optimization has been successful and is a proof of concept that the generated codon table using CUTE is successful and can be used for other synthetic hosts in the future.
Baylyi News-a information resource for working with Acinetobacter baylyi
Background:
While working with our project-building a synthetic toolkit for A. baylyi, we realized that while there are comprehensive information resources for other synthetic hosts and model organisms, there is no such information resource for A. baylyi. There is little known about the unique properties of A. baylyi like natural competency and ability to degrade aromatic compounds.
Problem:
During the course of the project, we had traversed multiple laboratory websites and small scattered information resources along with a vast multitude of research papers. It is quite difficult to stumble upon many of these scattered information resources without reading a large number of research papers. This might de-motivate many researchers from embarking on the use of A. baylyi for their research.
Our solution:
Seeing the potential of using A. baylyi and the large number of unique properties, we decided to create an information resource and host it on a free public server as www.baylyinews.weebly.com. The features of the website are minimal with basic information that we felt was very useful and referred to multiple times in our own experiments. Data includes commonly used strains of A. baylyi and vectors. We also included detailed data on vectors and laboratory pages on basic protocols that were helpful in our own experiments. Further, in an attempt to highlight the vast multitude of applications of A. baylyi as a host, we made a “Research in baylyi” section that includes a set of informative papers for each of the applications of A. baylyi.
Results:
The website was curated by members the bioprocess and metabolic engineering lab in the Department of Biotechnology in IIT Madras. These researchers work with A. baylyi on a regular basis and include our instructor and advisors. We received valuable insights in designing the structure of Baylyi News throughout the course of the project. We hope to connect more researchers and have created a form in the “Contact Us” section that allows visitors to send us queries. In the future, we hope to connect with the researchers working with A. baylyi around the world and help beginners troubleshoot their problems with ease.
Addition of A. baylyi data and other organism data onto ChassiDex
ChassiDex is an online information resource and database that equips one to work with unconventional synthetic hosts with ease. Having started work on this database from 2017, we faced challenges such as ensuring credibility of data and verifying the correctness of the codon table generator. Since we managed to verify the latter with the results of our project this year, we decided to work on the former.
Our contribution:
We sent out a google form across to researchers willing to add data through a google form. After that, we manually curated all the data and changed all the descriptive data to a collection of tags to reduce plagiarism and increase searchability through this markdown approach. Motivated by our preliminary successful results in our endeavours with A. baylyi, we decided to add our data and parts collection in the A. bayli section of ChassiDex.
Collaborations and future directions:
Our collaborative efforts using the portal on the iGEM page motivated Team Marburg that worked with V. natrigens, another unique host to add their data and comprehensive list of parts onto ChassiDex. We hope to publicize our efforts and collaborate with many more such teams with successful results through the interaction at Giant Jamboree 2018 while simultaneously maintaining the credibility of the data. Further, we will write about our results and initiatives in the form of a research article in the hope of motivating other researchers to contribute to and use our database.