Azospirillum Handbook
Azospirillum brasilense is an extremely beneficial rhizobacteria which can be the chassis for many agricultural projects. Handling Azospirillum is significantly different from E. coli in terms of the protocols. We were fortunate to have received proper guidance from our mentors who have worked on this chassis. There is a body of knowledge that is passed down orally from mentor to student in the Azospirillum research community that is often not documented in the literature. This cannot be accessed by anyone wishing to work with these niche model organisms. This makes our written guide, Azospirillum handbook a precious resource.
Adding a basic part and literature on it to the iGEM registry
exaA promoter
Pre-existing literature
The exaA promoter is present upstream to the quinoprotein alcohol dehydrogenase gene in A. brasilense sp7. The activity of this promoter is found to be induced significantly in the presence of glycerol, fructose, primary and secondary long chain alcohols(C4-C6). It has also been found that this is a σ54 dependent promoter. The -12/-24 site of the promoter is flanked by inverted repeats on both sides(IR1 and IR2) and it has been found that disrupting the IR2 site has adverse effect on the activity of the exaA promoter in its native system while disrupting IR1 does not show any significant effect. IR2 was found to be the binding site for the EraR response regulator. Using two hybrid assays it was confirmed that EraR physically interacts with the σ54 factor and enhances the promoter activity.
Experimental Data for exaA promoter activity
Pellicle formation experiments are a series of setups in which the aerotaxis movement of Azospirillum is studied under different mutations. Throughout our project, one of the hypotheses we wanted to check was if the alcohol inducible exaA promoter we have introduced into Azospirillum brasilense could be induced in hypoxia too. We thought we could check the above hypothesis qualitatively using this experiment. In this experiment, the final setup is done on a semi solid media in test tubes, where the oxygen levels gradually decrease at increasing depths. Our aim is to demonstrate that the Azospirillum can form a pellicle and express the exaA promoter at lower depths where hypoxic conditions are present. To know more, check out our Proof of Concept page!
Adding composite parts to the registry
FNR-UnaG composite part
The FNR promoter is a hypoxia inducible promoter of the the FNR (Fumarate and Nitrate Reductase) regulon naturally found in E. coli. The FNR protein is a transcriptional activator for a number of genes involved in anaerobic respiration. Azospirillum brasilense has nitrogenase genes that are regulated by the FNR/CRP complex and so it was hypothesized that this promoter would show regulated activity in Azospirillum.
To check this promoter’s activity, we needed a reporter gene that is functional under hypoxic conditions. The requirement of oxygen for the formation of the chromophore in fluorescent proteins like GFP and RFP renders them futile for experiments under hypoxia. The fluorescence of the UnaG protein, on the other hand, is not limited to aerobic conditions suggesting that it can be utilized for the vast expanse of experiments that require hypoxia. However, the presence of unconjugated bilirubin is a must for UnaG fluorescence. UnaG is a protein originally extracted from the muscle fibers of the Japanese eel, Unagi that utilizes bilirubin as a ligand for fluorescing. Hence, bilirubin has to be exogenously provided to the cultures.
Constructs like these can prove to be useful in synthetic biomedical engineering and would have wide uses in targeting specific tumor microenvironments. These can also be used for environmental project like ours that deal with low oxygen conditions.
T7 overexpression part for acdS
This part consists of the T7 promoter, the T7 RBS, the 1-aminocyclopropane-1-carboxylic acid deaminase gene (ACC deaminase/acdS), a 6x His tag fusion, and the T7 terminator. It overexpresses the acdS gene in a T7 overexpression system such as E. coli BL21 (DE3). It can be used to purify the ACC deaminase enzyme. To know more, check out our Parts page!
Standard curves of α-ketobutyrate
This year we are working on the ACC deaminase assay which uses spectrophotometry to determine the concentration of α-ketobutyrate which is the product of the reaction catalyzed by the ACC deaminase enzyme. This requires a standard absorbance curve of α-ketobutyrate which we were unable to find anywhere.
Therefore we have made a standard absorbance (540nm) curve of α-ketobutyrate by using its derivatised form as a colorimetric reading. This standard curve can be used as a reference by anyone performing the ACC deaminase assay to quantify ACC deaminase enzyme activity.
Climate Modelling
Our climate modelling module includes the generation of maps of India highlighting waterlogging hotspots. It is a significant contribution from our side because these maps are not readily available in literature
NDVI + NDMI map
NDVI + NDWI map
NDVI + MNDWI map
To know more, check out the climate modelling section in our Model page!
References
Azospirillum handbook
[1] https://www.genome.jp/dbget-bin/www_bget?gn:abf
[2] Oda Steenhoudt, Jos Vanderleyden, Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects, FEMS Microbiology Reviews, Volume 24, Issue 4, October 2000, Pages 487–506, https://doi.org/10.1111/j.1574-6976.2000.tb00552.x
[3] Gallori E. Bazzicalupo M. (1985) Effect of nitrogen compounds on nitrogenase activity in Azospirillum brasilense. FEMS Microbiol. Lett.28, 35–38.
[4] Oda Steenhoudt, Jos Vanderleyden, Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects, FEMS Microbiology Reviews, Volume 24, Issue 4, October 2000, Pages 487–506,https://doi.org/10.1111/j.1574-6976.2000.tb00552.x
[5] Zhulin IB, Bespalov VA, Johnson MS, Taylor BL. 1996. Oxygen taxis and proton motive force in Azospirillum brasilense. J. Bacteriol. 178:5199–5204
[6] Alexandre G, Greer SE, Zhulin IB. 2000. Energy taxis is the dominant behavior in Azospirillum brasilense. J. Bacteriol. 182:6042–6048
[7] Fukami, J., Cerezini, P., & Hungria, M. (2018). Azospirillum: benefits that go far beyond biological nitrogen fixation. AMB Express, 8(1). doi:10.1186/s13568-018-0608-1
Parts
[1] Lambden, P. R.; Guest, J. R.YR 1976. "Mutants of Escherichia coliK12 Unable to use Fumarate as an Anaerobic Electron Acceptor" Microbiology. 97 (2): 145–160. doi:10.1099/00221287-97-2-145
[2] https://www.uniprot.org/uniprotkb/A0A2K1G750/entry
[3] A Bilirubin-Inducible Fluorescent Protein from Eel Muscle Akiko Kumagai, Ryoko Ando, Hideyuki Miyatake, Peter Greimel, Toshihide Kobayashi, Yoshio Hirabayashi,Tomomi Shimogori and Atsushi Miyawaki. https://doi.org/10.1016/j.cell.2013.05.038
[4] Regulation of a Glycerol-Induced Quinoprotein Alcohol Dehydrogenase by σ54 and a LuxR-Type Regulator in Azospirillum brasilense Sp7. Vijay Shankar Singh, Ashutosh Prakash Dubey, Ankush Gupta, Sudhir Singh, Bhupendra Narain Singh, Anil Kumar Tripathi. https://doi.org/10.1128/JB.00035-17
