Climate change models are suggesting that the frequency and severity of heavy precipitation may increase in the near future all over the world—waterlogging is an unfortunate effect that climate change has on our planet. Global warming causes waterlogging to be even more unpredictable in its nature, making it troublesome to solve. Two million tonnes of food grain production are currently being lost annually due to waterlogging and this number is bound to increase.
Our team’s goal from the start was to help the poor farmers who are disproportionately affected by climate change, especially waterlogging. Our work does not stop with the iGEM competition. We are dedicated to making our work into an actual product which can create a difference in the farmers lives.
Future Production
We are going to form a startup named ‘Hydrazome’. The product that will be developed is described in Minimum Viable Product. The description of the production process is given below.
Azospirillum will be produced in batch cultures by large-scale liquid fermentation. For batch production in a fermentor, the key parameters to be controlled are :
a) Medium : We will be using BTB medium which contains (g/L): tryptone, 5 (Difco); yeast extract, 5, Na-gluconate, 5; NaCl, 1.2; MgSO4 ·7H2O, 0.25; K2HPO4 0.13; CaCl2 , 0.22; K2SO4 , 0.17; Na2SO4 , 2.4; NaHCO3, 0.5; Na2 CO3 , 0.09; Fe(III) EDTA, 0.07;
b) pH and Temperature: The pH will be adjusted to 7.0 after sterilisation. The optimum temperature is 36 ± 1 °C for the medium.
Formulated inoculant is practically the sole delivery vehicle of Azospirillum to the soil. Formulation is a critical issue for our inoculation system as introduced bacteria sometimes cannot find an empty niche in the soil. Therefore the inoculant must have the following three characteristics:
a) Support the growth of Azospirillum
b) Support the required number of viable cells in good physiological conditions for an acceptable period of time
c) Deliver enough Azospirillum at the time of inoculation required to obtain a plant response
We will be using the most popular formulation: Peat Inoculants.
The procedure of preparation of Peat Inoculants is:
For preparation of 100 g of inoculant for Azospirillum, 45g of ground peat (40 mesh) is thoroughly mixed with 5g of CaCO3 and 20 mL of tap water (final pH 6.8) stored in polyethylene bags sealed with a plug. The bags are sterilised (gamma-irradiated or tyndallization in an autoclave), and 30 mL of a 24 hour old bacterial culture (approximately 5 × 109 CFU/mL) are aseptically added to each bag, mixed, and incubated for an additional 7 days at 33 ± 2°C. Every 2 days, the peat is mixed by shaking the bags. The final number of bacteria in the inoculant range from 5 × 107 to 5 × 108 CFU/g of inoculant. The bags can be stored for several months at 4°C and a day before plant inoculation would be transferred to 30 ± 2°C for acclimation.
Future Testing
All the experiments performed yet have been performed in a controlled environment inside labs. We plan to hold field trials and quantify our results in open fields. We aim to test our Azospirillum in various field settings (waterlogged, normal, drought) , different types of soil (alluvial, red , black etc) and different crops (barley , rice , wheat). Some companies like Syngenta have even offered to assist us in field testing and we plan to collaborate with a large Agri-tech company and utilise its massive logistical network to aid our testing plans.
Dry Lab
In dry lab, we plan to extend our climate modelling to highlight drought hotspots in the country and also extend our waterlogging hotspot map from one state to the rest of the Indian subcontinent. Also, we plan to create a GSM for Azospirillum and perhaps work on plant metabolic modelling in the future. Another aspect of the future implementation of the dry lab will be to model if the modified Azospirillum has a tendency to outcompete the other soil bacteria if left unchecked.
Safety and Legal Aspects
Our Biocontainment Strategy:
There are many methods for biocontainment in genetic systems, but one of the most commonly used ones in the iGEM competition is the toxin-antitoxin system. Its theoretical simplicity and efficiency is what makes it a popular choice for either kill switch or biocontainment plans.
Toxin-Antitoxin (TA) systems are small genetic modules commonly found in prokaryotic genomes. A stable toxin and its labile antitoxin are usually the two components of this module. While toxins are always proteins, antitoxins can be either RNA or protein. Plasmid coded toxin-antitoxin systems are essential for post-segregational killing, also called as addiction.
There are many categories of TA systems. While choosing for our TA system, we carefully evaluated the pros and cons of each category. Toxin-antitoxin systems are typically classified according to how the antitoxin neutralises the toxin. In a type I toxin-antitoxin system, the translation of messenger RNA (mRNA) that encodes the toxin is inhibited by the binding of a small non-coding RNA antitoxin that binds the toxin mRNA. Even type III toxin-antitoxin systems involve the usage of RNA, such that the small fragment of RNA can bind directly to the toxin protein and inhibits its activity. But molecular biology experiments using RNA technologies require a lot more time to apply and expertise than that which could be afforded for only a module in the time frame of iGEM cycle. Hence, we decided to use type II systems where both the toxin and antitoxin are proteins. The toxin protein is inhibited post-translationally by the binding of an antitoxin protein.
One such toxin-antitoxin system is the rnlA and rnlB system found in E. coli K12 chromosome as an anti-phage mechanism. Under normal growth conditions, the rnlA toxin is inhibited by its cognate antitoxin, rnlB. However, rnlB disappears during T4 phage infections which renders the rnlA toxin, an endoribonuclease, active. rnlA cuts off phage RNA and prevents infection.lsoA-lsoB is a toxin-antitoxin system found in a cryptic plasmid pOSAK1 in E. coli O157:H7 which shares a weak homology with the rnlA-rnlB system.
Interestingly, the Dmd protein found in T4 bacteriophage directly binds to both rnlA and lsoA and inhibits their toxicity. Due the course of evolution, T4 bacteriophages have evolved this anti-toxin as a counter-defense mechanism against the bacterial toxin-antitoxin system.
The idea is to encode the lsoA/rnlA toxin in the artificially introduced plasmid along with the gene of interest and the Dmd anti-toxin in the genome of Azospirillum brasilense Sp7. This would mean that the plasmid of interest is fatal to the recipient soil bacteria while it does not affect the chassis.
The coding sequences of lsoA (ORF1) and lsoB (ORF2) in pOSAK1 overlap by 4bp and the secondary structure in this region seems to prevent translation of lsoB (ORF2) in the absence of co-translation of lsoA (ORF1). Hence, using the lsoA/rnlA toxin from E. coli and the Dmd anti-toxin from T4 bacteriophage is advantageous as they are from two different organisms.
Promoter Efficiency:
However, ensuring only the production of toxin and antitoxin will not fulfil the purpose effectively. A mechanism has to be set in place to ensure that the amount of antitoxin produced in the genome is enough to neutralise the toxin element produced in the plasmid. Since plasmids are more in number, the quantity of toxin elements produced in all would end up being more than that of antitoxin by the genome if the promoters are the same for both. This is where the promoter efficiency of both toxin and antitoxin elements comes into the picture.
Our plan was to first categorise promoters compatible in Azospirillum according to their efficiency and then model optimal matches of promoter efficiency of toxin, promoter efficiency of anti toxin, and copy number of plasmids such that the quantities of toxin -antitoxin produced are enough to neutralise each other.
However, Azospirillum is not a chassis organism that is characterised as well as that of E.Coli. While going through various repositories and papers we were not able to find enough constitutive promoters that were compatible in Azospirillum. Hence we could not go ahead with this plan of modelling and choosing promoters based on it.
However, the efficiency of promoters can be controlled indirectly using upstream activation sequences (UAS). An UAS is located upstream of the core promoter and serves as a binding site for specific transcriptional activators. While the core promoter is responsible for pre-initiation complex (PIC) recruitment and assembly, the UAS provides additional stability and regulation of PIC formation.
UAS’s with varying efficiencies could be used to control efficiencies of the promoter for both toxin and antitoxins too. We hope to carry the biocontainment plan forward by carrying out the wetlab experiments for this too.
Legal Aspects:
Now coming to the legal aspects, the first step will be to get the approval of GEAC and other equivalent state bodies in India as they will also do a risk analysis of our bioengineered product. Since our product is a biofertilizer, we have to abide by the Fertiliser Control Order of 1985 while deciding on the chemical composition and application in different soil conditions. The FCO order has certain regulations regarding the pricing of bio fertilisers and since we plan on commercialising the product, we need to do some ground-level market research to gauge the pricing of our biofertiliser.
EPS Production
Exopolysaccharides (EPS) are heteropolysaccharides secreted by many bacterial species such as Azospirillum brasilense, Bacillus subtilis, and even cyanobacterial species such as Phormidium ambiguum and Nostoc. They are typically produced in response to extracellular environmental stressors [1]. The chemical and physical properties of EPS vary depending on the organism as well as available nutrients. Species such as A. brasilense and P. ambiguum have been shown to promote soil aggregation as well as sequester nutrients and water [2]. The biofilm produced by EPS allows for microorganisms to remain in close proximity to each other, and with respect to A. brasilense this allows for better plant-bacterial interactions. EPS keeps the bacteria within the rhizosphere, allowing it to fix nitrogen easily and alleviate abiotic stresses of the plants. EPS has also been shown to perform heavy metal sequestration [3].
Taking all this into account, it is quite obvious why polymers based on exopolysaccharides are trying to be designed [4]. With respect to A. brasilense, one of the properties of EPS is its ability to absorb nutrients, water, and consolidate the soil. An issue such as waterlogging, where soil can be laden with water following which drought-like conditions are experienced, can be benefited by having such a polymer in its composition.
In Hydrazome, our aim was to provide a solution to plant stress responses during waterlogging as well as for the soil post-waterlogging. We will be overexpressing native A. brasilense genes (exoB, exoB2, and exoC) and comparing the nature of the EPS produced by them. We will also be comparing the production of EPS from EPS-related genes from cyanobacterial species. Our aim is to produce an exopolysaccharide of high viscosity; this will help with root associations, soil consolidations, and water absorption.
Drought and Salinity
While our project focuses on reducing waterlogging stress in plants, it has a huge potential of being used in drought and salinity conditions as well.
Drought is a severe issue in different parts of India and there have been a lot of studies and statistics which show that crop yields are severely affected every single year due to unpredicted drought conditions in different parts of the country. Drought causes the plant-water relation to be destabilised at cellular levels and plants respond to water shortages with severe morphological and physiological changes which may even end up with the plant dying.
However, it has been shown that ACC deaminase-producing bacteria, besides increasing root surface area to absorb more water and nutrients in the soil also can help the plant withstand drought stress. Our product, which uses genetically modified Azospirillum brasilense to help plants withstand waterlogging stress by breaking down ACC in the soil, can also be used in drought conditions.
Now coming to salinity - waterlogging conditions are often followed by soil salinity. Salinity stress is extremely harmful and happens to be one of the leading causes of poor crop productivity across the globe. Salt stress negatively impacts plants via the generation of reactive oxygen species (ROS) which act as a signal during salt stress simultaneously injuring plant roots and shooting tissue by disturbing enzymes, cell walls and membrane function. Similar to drought conditions being mitigated by bacteria producing ACC deaminase, salinity can also be tackled in the same way.
ACC deaminase helps plants ameliorate salt stress and since our projects deal with Azospirillum producing ACC deaminase, it can be used, with some modifications, to increase agricultural productivity in saline conditions.
References
[1] Inoculant Preparation and Formulations for Azospirillum spp. by Yoav Bashan and Luz E. de-Bashan
[3] Saikia, J., Sarma, R.K., Dhandia, R. et al. Alleviation of drought stress in pulse crops with ACC deaminase producing rhizobacteria isolated from acidic soil of Northeast India. Sci Rep 8, 3560 (2018). DOI: https://doi.org/10.1038/s41598-018-21921-w
[4] Nidhi Bharti, Deepti Barnawal. Chapter Five - Amelioration of Salinity Stress by PGPR: ACC Deaminase and ROS Scavenging Enzymes Activity. DOI: https://doi.org/10.1016/B978-0-12-815879-1.00005-7
[5] THE FERTILISER (CONTROL) ORDER 1985
[6] Massimiliano Marvasi, Pieter T. Visscher, Lilliam Casillas Martinez, Exopolymeric substances (EPS) from Bacillus subtilis : polymers and genes encoding their synthesis, FEMS Microbiology Letters, Volume 313, Issue 1, December 2010, Pages 1–9. DOI: https://doi.org/10.1111/j.1574-6968.2010.02085.x
[7] Chamizo, S., Adessi, A., Mugnai, G. et al. Soil Type and Cyanobacteria Species Influence the Macromolecular and Chemical Characteristics of the Polysaccharidic Matrix in Induced Biocrusts. Microb Ecol 78, 482–493 (2019). DOI: https://doi.org/10.1007/s00248-018-1305-y
[8] Pratima Gupta, Batul Diwan, Bacterial Exopolysaccharide mediated heavy metal removal: A Review on biosynthesis, mechanism and remediation strategies, Biotechnology Reports, Volume 13, 2017, Pages 58-71, ISSN 2215-017X. DOI: https://doi.org/10.1016/j.btre.2016.12.006.
[9] Programmable Synthetic Upstream Activating Sequence Library for Fine-Tuning Gene Expression Levels in Saccharomyces cerevisiae. Shiyun Li, Lizhou Ma, Wenxuan Fu, Ruifang Su, Yunying Zhao and Yu Deng. DOI: https://doi.org/10.3390/toxins9010029.
[10] RnlB Antitoxin of the Escherichia coli RnlA-RnlB Toxin-Antitoxin Module Requires RNase HI for Inhibition of RnlA Toxin Activity. Kenta Naka, Dan Qi, Tetsuro Yonesaki, Yuichi Otsuka. DOI: https://doi.org/10.1016/j.btre.2016.12.006.
[11] Toxin–antitoxin systems: why so many, what for? LaurenceVan Melderen. DOI: https://doi.org/10.1016/j.mib.2010.10.006.
[12] A Primary Physiological Role of Toxin/Antitoxin Systems is Phage Inhibition. Sooyeon Song and Thomas K.Wood. DOI: https://doi.org/10.3389/fmicb.2020.01895.
[13] Dmd of bacteriophage T4 functions as an antitoxin against Escherichia coli LsoA and RnlA toxins. Yuichi Otsuka, Tetsuro Yonesaki. DOI: https://doi.org/10.1111/j.1365-2958.2012.07975.x.
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