Team:IISc-Bangalore/Contribution
Contribution
A Novel Plug-n-Play Bioremediation Platform
Quite a number of iGEM teams have worked with organophosphate bioremediation in the past. For the sake of future teams willing to work along the same line, we provide a brief description of their works below:
| Serial No. | Team | Year of participation Project | objective |
|---|---|---|---|
| 1 | SCAU-CHine | 2013 | Membrane display of OpdA using ice nucleation protein in order to degrade parathion; Building biosensor to quantify the amount of degradation product p-nitrophenol (PNP); Used various microbial enzymes (e.g. NphA1, NphA2 etc.) to achieve degradation of p-nitrophenol |
| 2 | University of Texas at Austin | 2016 | Employed metabolic pathways to degrade sarin, glyphosate and paraoxon into PNP; Utilized beta-ketoadipate pathway to achieve conversion of PNP into food sources for bacteria |
| 3 | LN-Shiyan-China | 2016 | Wanted to use E. coli as a OpdA-synthesizing bioreactor to be released into OP-polluted substrat |
| 4 | SSTi-SZGD | 2017 | Utilized LightOff optogenetics system to improve the microbial method for carbendazim (MBC) residue degradation in soil involving MheI enzyme |
| 5 | Emory BioTech | 2018 | Utilized mutants of ArPTE and PoOPH enzymes to save benign flies from malathion |
| 6 | XMU-China | 2020 | Used glyphosate oxidase to convert glyphosate into AMPA and glyoxylate; Based on NADPH read-out, built an indirect biosensor for glyphosate |
Drawing inspiration from all these works, and depending on our understanding as well as hard work, we have developed CellOPHane. It is a low-cost, modular, novel, safe, plug-n-play filtration-bioremediation platform. We have used OpdA as the functional partner in C2 protein. In future, another team can just replace OpdA with some other enzyme and can use the same C1 protein as us to develop a bioremediation system effective against some other pollution. In that sense, the variety of such systems is restricted only by the number of known, effective hydrolases. It can even be utilized to immobilize microplastics, heavy metal etc. Thus, our design is truly universal and powerful.
For more details, check our Description, Design, and Implementation pages.
Hardware
Our hardware endeavours share the same goal of making an impact. We have progressed quite a bit towards building our functional prototype.
CellOPHane is a good example of how materials engineering can be integrated in a synthetic biology project. We have deduced an optimal solvent system (DMSO+PVA) which can be used by future iGEM teams to make MCC sheets which can serve many purposes in their respective projects.
For more information, visit the Hardware section under the Design page.
Parts
During the course of our project, we utilized, developed, and optimized a number of genetic sequences. These are the fruits of the engineering design cycle we iterated in relation to our wetlab works. The parts are tabulated below along with proper classification:
New Basic Parts| Serial No. | Name Part | Number |
|---|---|---|
| 1 | SpyTag002 | BBa_K3765000 |
| 2 | SpyCatcher002 | BBa_K3765001 |
| 3 | Glycine-Serine linker (GSGGS) | BBa_K3765005 |
| 4 | Glycine-Serine linker (GSSGS) | BBa_K3765006 |
| 5 | OpdA Signal Peptide | BBa_K3765007 |
| 6 | 22 Amino Acid Linker | BBa_K3765009 |
Improved Basic Parts
| Serial No. | Name | Part Number |
|---|---|---|
| 1 | PelB Signal Peptide | BBa_K3765002 |
| 2 | 10xHis Tag | BBa_K3765003 |
| 3 | dCBD (Double Cellulose Binding Domain) | BBa_K3765004 |
| 4 | OpdA | BBa_K3765008 |
| 5 | sfGFP (Superfolder GFP) | BBa_K3765010 |
New Composite Parts
| Serial No. | Name | Part Number |
|---|---|---|
| 1 | PelB_Signal+10xHis+SpyCatcher002+dCBD | BBa_K3765012 |
| 2 | OpdA_Signal+OpdA+22aaL+SpyTag002+sfGFP+10xHis | BBa_K3765013 |
| 3 | OpdA_Signal+OpdA+22aaL+SpyTag002+10xHis | BBa_K3765014 |
| 4 | OpdA+22aaL+SpyTag002+10xHis | BBa_K3765015 |
| 5 | T7 RBS + SpyCatcher002+dCBD | BBa_K3765016 |
| 6 | T7 RBS + OpdA+SpyTag002+sfGFP with OpdA signal peptide | BBa_K3765017 |
| 7 | T7 RBS + OpdA+SpyTag002 with OpdA signal peptide | BBa_K3765018 |
| 8 | T7 RBS + OpdA+SpyTag002 without OpdA signal peptide | BBa_K3765019 |
| 9 | SpyCatcher002+dCBD under T7 promoter | BBa_K3765020 |
| 10 | OpdA+SpyTag002+sfGFP with OpdA signal peptide under T7 promoter | BBa_K3765021 |
| 11 | OpdA+SpyTag002 with OpdA signal peptide under T7 promoter | BBa_K3765022 |
| 12 | OpdA+SpyTag002 without OpdA signal peptide under T7 promoter | BBa_K3765023 |
For more information on these Parts, visit the Parts page.
Human Practices
Our team has focussed consistently on integrating the views of various stakeholders into our project as a part of our IHP journey. We have followed the human-centred design process propounded by iGEM Calgary 2019 and have strived to emphasize the mutualistic relationship of science and society. In doing so, we have also tried to underline a rational and systematic approach to human practices. We have tried to adopt standard systematic methodologies of research in social sciences and business administration to identify our stakeholder. Furthermore, for the ease of future iGEM teams, we have laid down a set of 'actionables' for each of the steps that iGEM Calgary enunciated in the human-centred design cycle. We recognize that the HP plans for a specific team are highly project-centric. We have however tried to lay down a more concrete set of pointers for every step in their HP journey for teams to reflect upon. We believe this would be of great help to future iGEMers in their project design and in their appreciation of societal implications of their project. For more details, visit our Human Practices page
Wiki Development
As must be evident from our wiki, we have tried to stick to the art of minimalism. Not just on our wiki, "less is more" has been a guiding principle for all our design-related activities. Many previous iGEM wikis, including ones which have been rewarded, are extensively animation-heavy and have precipitously large loading times, which reduces their accessibility, particularly in developing countries with not-so-good Internet facilities. We have tried to change this, and we believe that the extensive documentation provided on our wiki despite the minimalistic design shall be an inspiration for future teams. We have also rigorously stuck to the 'three click rule', and incorporated features to improve accessibility and navigability like introductory abstracts on every page, a "Back to Top" button and so on. Besides these, here are some other contributions made by our team to wiki development -
- Bootstrap happens to be the popular choice among high quality wikis of iGEM. However, we abandoned Bootstrap in favour of MaterializeCSS, a relatively lightweight and simple framework.
- MaterializeCSS and AnimateOnScroll are the only frameworks we have used on our website, in addition to inbuilt MathJax and JQuery. We hope to achieve a decrease in loading times.
- Although iGEM wikis are judged based on how they look on desktops, we could not ignore the fact that most internet users use mobiles. Keeping this in mind, we have also constructed a fully usable, mobile version of our wiki.
- Preloaders serve to assure the user that something is indeed happening behind the hood, and nothing has gone amiss. In addition to realising the importance of preloaders, we utilised a simple preloader animation to reduce overall workload.
- We have uploaded all our code and assets on a GitHub Repository. Users can download all of this with a single click and view them in their editor of choice. Users are free to contact our wiki developer (Chandan R.T.).
- We used iGEM WikiSync, developed by 2020 BITS Pilani - Goa team. This allowed us to code in our editor of choice. iGEM-wikisync itself is an improvement on wiki generators made by several iGEM teams in the past. However, iGEM-wikisync does not allow us to utilize Template pages (at least not to our knowledge). Therefore, synchronizing common elements like navbar and footer across multiple pages became a problem. We developed a simple python script for this purpose, named "injection.py" and can be found in our repository.
Troubleshooting Tips
We are of the opinion that our wetlab work went well. In the short time in the lab amidst the pandemic, we were able to clone our constructs and express our proteins of interest successfully. The Gibson Assembly was an interesting new method to learn, and we executed it quite perfectly. We also progressed significantly in our drylab endeavours.
However, we would like to share some troubleshooting tricks that we hope we knew when we had been working in the labs.
- A lot of thoughts should be put into the primer design. Any chance of primer dimer formation should preferably be eliminated.
- If the protein of interest is not hydrophilic enough, one might have to remove the signal peptide (mostly hydrophobic), if any, provided the peptide is not absolutely essential for obtaining the protein.
- Prior to transforming plasmid into the expression host, one must go through literature to obtain clarity about the essential expression conditions required by the protein of interest. The protein might need metal ions or some small molecules for proper folding itself (e.g., OpdA required Co(II) ions)!
- If the protein of interest is a metallozyme, it is better not to use metal-based pulldown process (e.g., Ni-NTA beads). Use of alternative processes like immunoprecipitation is advised.
- While growing bacteria in some general media without any antibiotic (like sorbitol media), one should stringently follow aseptic techniques. The risk of contamination is pretty high in this case.
- Before using a chemical substance in some experiment, its MSDS should be studied carefully in order to know about its physical properties (like degradation temperature, solubility etc.). This practice would be of high help in experimental design and would pave the way for experimental success