Aim
Our project Hydrazome, was developed so that we could help prevent the damage caused to crops because of the release of harmful amounts of ethylene during waterlogging. And so, our project’s ideal and theoretical aim would be to reduce the levels of ethylene released by crops during a waterlogged state.
The Role of Azospirillum
In this iGEM cycle, our team has used Azospirillum as the ‘vehicle’ that will enact the role of the biofertiliser here - it will take up the excess ACC exuded out by the roots of the plant, and break it down into ammonia and α-ketobutyrate - with the help of the enzyme ACC Deaminase. Azospirillum’s expression of ACC Deaminase will be regulated such that it only occurs under hypoxic conditions such as those induced by waterlogging.
Objectives to Address
We plan to address these four objectives through our project to provide for an exhaustive proof of concept at the molecular biology level:
- To regulate acdS expression so as to limit it to hypoxic conditions
- To confirm that Azospirillum is taking up ACC
- To determine the rate of the exudation of ACC by plants
- To determine the rate at which ACC Deaminase breaks ACC down
We have laid out the plan to address the above objectives in the following tabs. We have also added our successes within those categories.
Need for acdS regulation
Regulating the expression of our acdS gene so that it produces ACC Deaminase only during the second peak of ethylene production, has been an important aspect of our project. Since hypoxic conditions set into the surrounding in a delayed fashion, we thought that it would serve as an appropriate inducer for our gene. Hence cloning the hypoxia induced promoters and checking for their efficiency is a integral step for the proof of concept.
Pellicle Experiment
Aim
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 have wanted to check was that 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 hypoxic conditions gradually increase 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.
Plan
We set up inoculations of Azospirillum transformed with the pCZ plasmid containing the exaA promoter as our “experiment”, as well as Azospirillum transformed with pCZ plasmid as our “control”. We grew both the constructs in NFB(-) media (Nitrogen free broth without added nitrogen sources) and NFB(+) (Nitrogen free broth with added nitrogen sources) media.
Downstream to the exaA promoter, the pCZ plasmid contains the lacZ gene. Our aim was to qualitatively characterize exaA expression through the expression of lacZ. In order to do this, we added X-gal to the setups of both the constructs in both the media. The X-gal gets broken down by the β-galactosidase enzyme coded by the lacZ gene, and results in a blue colour.
In NFB(-) media, the nitrogen fixing Azospirillum has to obtain its own nitrogen, which it does using the Nitrogenase enzyme. However, this enzyme works only in anaerobic environments, and for that, the pellicle should be formed at a lower depth in the semi solid media. If the exaA promoter is active under hypoxic conditions, we would expect that the colonies of Azospirillum containing the pCZ-exaA construct grow below the surface and show lacZ expression by displaying a blue colour.
In NFB(+) media, Azospirillum receives nitrogen, and hence it does not need to go to a lower depth to grow and form pellicles. This enables Azospirillum to grow in aerobic conditions. Hence, we used this setup as a control to compare exaA activity in aerobic conditions with the hypoxic one. If the exaA promoter is active in aerobic conditions, it is expected to show lacZ expression in this setup.
In order to ensure that the difference in expression of lacZ gene was not due to the difference in cell growth, we decided to check it with TTC (Triphenyl tetrazolium chloride), a redox indicator commonly used in biochemical experiments to indicate cellular respiration [1]. The presence of red colour indicates that respiration has taken place, indirectly implying cell growth has occurred. Ideally we would expect the setups containing both constructs to have similar intensity or thickness of red colour in both types of media.
Results Obtained
TTC:
It was visually interpreted that the intensity of the red colour was similar in both the NFB(+) and NFB(-) media for both the constructs (pCZ-exaA and pCZ). This implies that the respiration rate and hence, the growth in all the four test tubes were more or less the same. We can conclude that the difference in growth does not contribute to the difference in expression levels of lacZ.
X-gal:
It was observed that all the four test tubes showed blue colour. This is likely due to the fact that the hypothesised promoterless pCZ vector shows leaky lacZ expression (implying the presence of a promoter upstream to the gene). However, in NFB(-) media, it was visually apparent that the test tube with pCZ-exaA constructs had a higher blue colour intensity than the one with only pCZ. This might be due to exaA promoter activity in hypoxic conditions. This is a crude qualitative estimation and hence, the derived conclusions are not definite.
Miller's Assay
Aim:
Qualitative experiments, like the pellicle set up experiment, help us to verify that our constructs work under the conditions we had hypothesised them to. But in order to characterise them and obtain data about the construct’s utility, it is necessary to perform quantitative experiments as well.
Plan
Hypoxic Environment:
Before proceeding with the necessary assays, we had to come up with a way to create varying levels of hypoxic conditions, so that we could perform the assays in them. We decided to use sodium sulphite in our experiments, to generate hypoxia.
By changing the concentration of sodium sulphite in the media solution, we could obtain different ranges of dissolved oxygen in it (measured in ppm). We measured these ranges using a Dissolved Oxygen (DO) meter.
Image of the DO meter we used [2]
We first set off to create a standardisation curve of sodium sulphite concentration versus the hypoxia it generates (measured in dissolved oxygen in ppm). We created sodium sulphite concentrations ranging from (0g/L) to (1g/L), in minimum malate media (the media we used to carry out the experiment). Then we measured the DO in regular intervals of time in each setup.
After carrying out the standardisation measurements, we realized that our instrument’s least count was higher than the range we wanted to create the hypoxic environment gradient in. Also, the time taken by the instrument to measure a reading was extremely high. Due to the above technical constraints, we realised that we could not rely upon the data we collected for the standardisation curves. It then struck us, that one of the main ideologies of iGEM is to share knowledge and data to improve each others' project. We found out that the 2019 iGEM NEFU_China team had also generated a standardisation curve of sodium sulphite versus the dissolved oxygen, and this helped us to use the data for our further experiments. Their data points:
Dissolved oxygen changed curve under a series of concentrations of sodium sulphite (0, 0.25, 0.5, 0.75 and 1 g/l Na2SO3) [3]
We also had to determine that sodium sulphite itself is not toxic for the growth of Azospirillum and does not interfere with its metabolism. We checked this by comparing growth curves of wild type Azospirillum in LB and wild type Azospirillum in LB+ Sodium Sulphite. To know more about this experiment, visit our Experiments page.
Assay
β-Galactosidase, is a protein that is encoded by the lacZ gene which is present downstream to our exaA promoter. The protein’s function is to cleave lactose into glucose and galactose, so that they can be used as energy sources.
Breaking down of lactose by β-galactosidase [4]
β-galactosidase can also recognise o-nitrophenyl-β-D-galactoside (ONPG) as a substrate in place of lactose. It gets cleaved into galactose and o-nitrophenol, which has a yellow colour. In order to quantitatively determine the expression of exaA promoter in varying hypoxic conditions, we decided to perform the Miller’s Assay, where ONPG is added to the growth media.
Breaking down of ONPG by β-galactosidase [5]
β-galactosidase activity is quantified through the absorbance of the yellow colour generated by o-nitrophenol in Miller’s units. We planned on quantitatively comparing the expression of exaA promoter upstream to lacZ gene using Miller’s units in varying hypoxic environments (using the appropriate amount of sodium sulphite based on the standardisation curves)
Expected Results
We were expecting to observe a varying range of β-galactosidase activity of the lacZ gene for different hypoxic levels. Some hypoxic levels would have higher amounts of expression. Using growth curve results, it should be checked that Azospirillum can survive considerably, in those hypoxic levels.
Observations
Due to time and technical constraints, we were unable to carry out the Miller’s assay. However, we plan to do the same in the future and strengthen our proof of concept.
Fluorescence Assay
Aim
To check the activity of the exaA promoter under alcohol induction in E. coli.
Plan
exaA is an alcohol inducible promoter in Azospirillum and we wanted to check if it works as an alcohol inducible promoter in E. coli as well. E. coli has very good fermentative properties so our hypothesis was that if the promoter is alcohol inducible in E. coli then it should be hypoxia inducible as well. We felt that this would be important because introducing a new hypoxia inducible promoter in E. coli can later help in cancer research and it would be a contribution from our side.
We received a construct of exaA with lacz as a reporter construct in pCZ from BHU (Banaras Hindu University). We tried doing blue white screening using X-gal but ended up getting colonies in both alcoholic conditions(with glycerol) and control(without glycerol). We learnt about the possibility of leaky expressions of lacZ. Also, Dr. Alexandre Gladys pointed out to us that lacZ may not fold properly in hypoxic conditions which could affect our results. Instead, we were recommended that mCherry would be a better reporter gene to use as it is stable in hypoxic condition. We verified the same by looking into some literature as well [6]. Hence, we decided to change our reporter to mCherry and aimed to make a construct of exaA with mCherry in the pET15b vector. However, the restriction sites that are flanking our exaA promoter were not present in the pET15b vector such that exaA can be cloned upstream of mCherry. We couldn't introduce new sites in exaA because we were replacingthe T7 promoter and restriction enzymes either didn't flank that region or the required restriction enzymes were not available. Therefore, we decided to make our construct through RF Cloning. After making the construct we aimed to check for the promoter activity under alcoholic induction through mCherry fluorescence assay [7].
Assay
Transform the RF clones into DH5α chemical competent cells in LBA+Amp plates.
Inoculate colonies in 10ml LB+Amp at 37°C for 14hrs.
For secondary inoculation do it in duplicate in 10ml M9 minimal media each. Once the OD reaches 0.7, add glycerol in one of the test tubes and incubate them at 37°C for 6hrs.
Pellet down the cells at 8000rpm for 2minutes. Lyse the cells on ice for 15 minutes using 50μL of RIPA lysis buffer [10mM Tris-HCL, 50mM NaCl, 1mM Na-orthovanadate, 30mM Na-pyrophosphate, 50mM NaF, 1% Nonidet P-40, 0.1% SDS, 1mM PMSF, 1% Triton X-100, 0.5% Na-deoxycholate, and dissolved protease inhibitor cocktail (Sigma Aldrich) in water, pH 7.4].
Centrifuge the cells at 15,000 rpm for 5 minutes and dilute 20 μL of the cellular lysate 1:10 in water. Add 180μL of the diluted cellular lysate to a black 96-well plate to eliminate cross contamination of the fluorescent signal from adjacent wells.
Record the mCherry fluorescent signals. The mCherry standard curve can be obtained using purified mCherry protein by serially diluting (range of 10 pg to 1 μg) in OPTI-MEM (Invitrogen).
Expected Observations
Fluorescence should be observed only where the cells were grown in glycerol because our exaA promoter is alcohol inducible. In the absence of alcohol, mCherry expression should not happen as the exaA promoter should not get induced. But due to time constraints we were not able to perform this assay. However we definitely plan to do so in the future.
References
[1] Witty M. (2012). The process for 2, 3, 5 - triphenyl – tetrazolium chloride synthesis, an intellectual property seized immediately after world war II. Bulletin for the History of Chemistry 37(2):91-95.
[2] http://lutron1976.myqnapcloud.com/database/pdf/DO-5509.pdf
[3] https://2019.igem.org/Team:NEFU_China/Results
[4] https://www.sigmaaldrich.com/IN/en/product/sigma/g5635
[5] https://microbeonline.com/onpg-test-galactosidase-principle-procedure-results/
[6] Wang Y, Wang H, Li J, Entenberg D, Xue A, Wang W, Condeelis J. Direct visualization of the phenotype of hypoxic tumor cells at single cell resolution in vivo using a new hypoxia probe. Intravital. 2016;5(2):e1187803. https://doi.org/10.1080/21659087.2016.1187803
[7] Duellman T, Burnett J, Yang J. Quantitation of secreted proteins using mCherry fusion constructs and a fluorescent microplate reader. Anal Biochem. 2015 Mar 15;473:34-40. https://doi.org/10.1016/j.ab.2014.12.010
Aim
Growth curves are the empirical models of the evolution of a particular quantity with time. In molecular biology, these aid in checking the metabolic load of the chassis organisms with additional inserts as compared to the wild type version. We realized that for our project, growth curves could also be a great way to analyse the expression of our gene through the utilisation of the substrate in various conditions with control and experimental setups.
Plan
We first decided to set up growth curves of the below strains in Minimal Malate media in NFB(-), to obtain control growth curves in the absence of other agents or substrates:
1) Wild type Azospirillum brasilense
2) Wild type Azospirillum oryzae
3) Azospirillum brasilense with pCZ-exaA construct
4) Azospirillum brasilense with pCZ-exaA-acdS construct
We then decided to set up growth curves of the below strains in Minimal media + glycerol in NFB(-) to confirm expression of exaA promoter under glycerol induction.
1) Wild type Azospirillum brasilense
2) Azospirillum brasilense with pCZ-exaA construct
3) Azospirillum brasilense with pCZ-exaA-acdS construct
Obtaining growth curves of the below strains in Minimal media + glycerol + ACC would help us to confirm the expression of acdS when exaA is induced by glycerol.
1) Azospirillum brasilense with pCZ-exaA construct
2) Azospirillum brasilense with pCZ-exaA-acdS construct
Obtaining growth curves of the below strains in Minimal Malate media + sodium sulphite (SS) + ACC would help us to confirm the expression of acdS if/when exaA is induced by sodium sulphite
1) Wild type Azospirillum brasilense
2)Azospirillum brasilense with pCZ-exaA construct
3)Azospirillum brasilense with pCZ-exaA-acdS construct
Obtaining growth curves of the below strains in Minimal media + glycerol + sodium sulphite + ACC would help us to confirm the expression of acdS in the presence of both glycerol and hypoxia generated by sodium sulphite
1) Azospirillum brasilense with pCZ-exaA construct
2)Azospirillum brasilense with pCZ-exaA-acdS construct
Expected Results
Without SS nor ACC:
Growth of Azospirillum oryzae to be slower/lesser than Azospirillum brasilense.
Growth of the rest of the Azospirillum brasilense constructs should be more or less similar to the wild type of Azospirillum brasilense, as the inducers of the promoters are absent. Hence the inserted gene will not be able to cause any additional metabolic load.
With Glycerol, Without SS, Without ACC:
Wildtype Azospirillum brasilense’s growth curve under glycerol would give data about toxicity/impact of glycerol on the growth as a chemical in general when compared to the growth of wildtype Azospirillum brasilense in the absence of glycerol.
In glycerol, exaA would be expressed. Hence, it could be expected that the growth of Azospirillum brasilense with pCZ-exaA construct and Azospirillum brasilense with pCZ-exaA-acdS construct would be similar to each other and lower than the wild type due to the metabolic load of the promoter.
With SS, Without ACC
Wildtype Azospirillum brasilense’s growth curve under sodium sulphite would give data about toxicity/impact of glycerol on the growth as a chemical in general when compared to the growth of wildtype Azospirillum brasilense in absence of sodium sulphite.
It is being checked if exaA expresses itself in the hypoxic conditions created by sodium sulphite. Hence, it could be hypothesised that the growth of Azospirillum brasilense with pCZ-exaA construct and Azospirillum brasilense with pCZ-exaA-acdS construct would be similar to each other and lower than the wild type due to the metabolic load of the promoter.
With glycerol, Without SS, With ACC
In the presence of ACC, the acdS gene should be utilised and hence expressed. Azospirillum brasilense with pCZ-exaA-acdS construct‘s growth curve should ideally be lesser/slower than that of Azospirillum brasilense with pCZ-exaA construct, due to the metabolic load of the acdS gene.
With SS, With ACC
If exaA is induced in hypoxic conditions, created by sodium sulphite, the acdS gene should be utilised and hence expressed, in the presence of ACC. Azospirillum brasilense with pCZ-exaA-acdS construct‘s growth curve should ideally be lesser/slower than that of Azospirillum brasilense with pCZ-exaA construct, due to the metabolic load of the acdS gene.
With SS, With Glycerol, With ACC:
Although the individual impacts of glycerol and sodium sulphite have been determined, wildtype Azospirillum brasilense's' growth curve under both sodium sulphite and glycerol would give data about toxicity/impact of both of their presence on the growth, and would help us check if the toxicity levels are additive or not.
Similarly we can check if the expression of exaA and acdS under glycerol and sodium sulphite is additive or if the presence of one impacts the inducibility of the other.
Observations
We set up growth curves of Azospirillum brasilense wild type and the modified ones in Minimum Malate Media with NFB(-) conditions, but only the wild type grew. pCZ exaA-acdS showed growth after several days, but we could not confirm if it was contamination or the growth of our chassis. One of the hypotheses we formed out of this observation was that the metabolic load of the exaA-acdS construct on Azospirillum is higher than what it can withstand with the given media (i.e one of the chemicals may be a limiting factor for its growth). We could check this out by increasing the quantities of every chemical in the Minimal media one by one. Due to time constraints, we could not carry this out further.
Aim
The aim of this module was to investigate the diffusion of ACC from out of the roots of the plant into the bacteria. As part of our Partnership - you can read more about it on our Partnership page - the UBC iGEM team prepared an experimental protocol which is as follows:
The above PDF displays the protocol for an experiment designed by the UBC iGEM Team, as part of the many things that our teams partnered on. The experiment allows one to assay ACC levels and ethylene levels released by plants under different conditions.
Aim
The purpose of this assay/module is to use an in vitro biochemical assay to ascertain the enzymatic activity of ACC Deaminase (acdS). Two essential components are necessary for the assay: (1) the enzyme ACC Deaminase and (2) the substrate ACC.
Plan
The ACC Deaminase assay that we planned to perform was taken from Penrose and Glick 2003 [4], which in itself is a modified version of Honma and Shimomura 1978 [2]. The assay is based on the quantitative estimation of 𝛂-ketobutyrate through the absorbance at 540 nm of its 2,4-dinitrophenylhydrazine derivative. A standard curve is first made with the absorbance values at 540nm of the 2,4-DNP derivatives of known concentrations of 𝛂-ketobutyrate.
We plan to incubate the crude cell extract (which contains the ACC Deaminase protein) along with ACC, for different concentrations of ACC (rainging from 0.1M to 0.5M). This data would give us the increase in 𝛂-ketobutyrate production with respect to ACC concentration, which further gives insight into the enzyme kinetics of ACC Deaminase.
(1) Obtaining the enzyme ACC Deaminase
Inspired by the work and results from Farajzadeh et. al 2009 [1], we decided to use the ACC Deaminase gene from Pseudomonas fluorescens. We had the acdS gene synthesised from Synbio Technologies in a pUC57 backbone.
Using primers, we amplified the acdS gene through a PCR reaction. More about it can be found on our Engineering page.
The acdS gene was cloned into the pHis17 backbone using restriction-free cloning. The pHis17 system consists of (in order): a T7 promoter, a T7 RBS, the acdS gene, a T7 terminator. This system was transformed into E. coli BL21(DE3) cells which can express the T7 RNA Polymerase under IPTG induction.
(2) Obtaining ACC (the substrate) and 𝛂-ketobutyrate (the product)
We purchased ACC and 𝛂-ketobutyrate in powdered format. We then prepared stock solutions of 0.5M ACC in water and 100mM 𝛂-ketobutyrate in 0.1M Tris-HCl pH 8.5.
(3) Preparing standard curve for 𝛂-ketobutyrate
We were able to generate the following standard curve for 𝛂-ketobutyrate:
The above standard curve correlates the concentration of 𝛂-ketobutyrate with the observed absorbance of their 2,4-DNP derivatives. The correlation can then be used to estimate solutions with unknown 𝛂-ketobutyrate concentrations.
You can read more about the protocol for generating the standard curve here.
Selection, confirmation of cloning, and an SDS-PAGE was performed to check the size of the protein. See more in our Engineering page.
Expected Results
(1) Cloning of acdS into pHis17
- The expected plasmid clone we wish to obtain has a map that looks like this:
The above plasmid map illustrates the pHis17-acdS clone we wish to engineer. The T7 promoter, T7 RBS, the acdS gene, the 6x Histidine tags and the T7 terminator can be seen clearly.
- Amplification of acdS from pUC57-acdS should give us a band of size ~1 kb
- The primary PCR to generate the megaprimer should yield a PCR product that is present in a band of size ~1 kb
- The secondary PCR should yield a product that is present in a band of size ~3.7 kb
- The transformation should yield a larger number of colonies in the Test plate (containing the clone) than in the Vector Control plate (containing the backbone)
- A PCR reaction with acdS-specific primers should yield ~1 kb bands with the clone
- Sequencing of the cloned plasmid should show 100% sequence identity with the acdS sequence
(2) Expression of the acdS protein
An SDS PAGE run for the cell lysate of E. coli BL21(DE3) containing pHis17-acdS is expected to give a band ~37 kDa corresponding to the acdS-His tag construct.
(3) ACC Deaminase Assay
The acdS assay is expected to give a pattern of 𝛂-ketobutyrate that increases with ACC concentration, and plateaus after a certain point. The absorbance data obtained from the 2,4-DNP derivatives of the cell lysates, which are incubated with varying ACC concentrations, will give us corresponding absorbance values. The values will be plugged into the standard curve to obtain molar concentrations of 𝛂-ketobutyrate produced at varying ACC concentrations. This data will then be plotted to give us an idea of the speed with which ACC deaminase breaks ACC down into 𝛂-ketobutyrate.
Results Obtained:
(1) 1% Agarose Gel after PCR amplification of acdS from pUC57-acdS
It can be seen that the three bands in the replicate wells containing the PCR amplified product, lie parallel to the 1 kb band, which corresponds to the 1 kb size of the acdS gene.
(2) 1% Agarose Gel after Primary PCR to generate pHis17-acdS overlap megaprimer
It can be seen that the four replicate bands of the primary PCR products lie along the 1 kb band expected for the pHis17-acdS megaprimer. However, the appearance of a distinct but nevertheless significant band in the negative control came unexpectedly.
(3) 1% Agarose Gel after Secondary PCR to complete the cloning of acdS into pHis17
It can be seen that the Test well shows a band corresponding to 3.7 kb as expected for the pHis17-acdS clone. The Vector Control well shows no bands as expected.
(4) Plates after transformation of cells after DpnI digestion of secondary PCR products
Colonies were observed in both the Test and Vector Control plates. This was not expected; however, since the colonies appeared distinct in both plates, we decided to proceed with our protocol and do confirmatory checks later.
(5) 1% Agarose Gel after Confirmatory PCR using acdS-specific primers on pHis17-acdS
It can be seen that all 5 wells (corresponding to 5 colonies picked) show amplification of acdS (1 kb) as expected from the confirmatory PCR.
(6) 12% SDS-PAGE Gel after gradient concentration IPTG induction for T7-based expression of acdS
It can be seen that a dark band corresponding to ~38 kDa is present in both the induced and uninduced wells. It is as expected for the ACC Deaminase protein.
With respect to the ACC assay, we performed it according to the protocol used by Farajzadeh et. al 2013[1], with modifications to account for the use of the T7 system. However, the results were not as we expected. This led us to believe that there might have been some issue with the RF cloning of acdS into pHis17.
Conclusion
While the PCR confirmation and sequencing results tell us that the acdS gene was cloned successfully into pHis17, the lack of a proper expected result from the ACC Deaminase Assay suggests that we need to look into more checks for cloning, and troubleshooting the appearance of mixed results.
References
[1] Farajzadeh D, Aliasgharzad N, Sokhandan Bashir N, Yakhchali B. Cloning and characterization of a plasmid encoded ACC deaminase from an indigenous Pseudomonas fluorescens FY32. Curr Microbiol. 2010 Jul;61(1):37-43.https://doi.org/10.1007/s00284-009-9573-x10.1007/s00284-009-9573-x
[2] Mamoru Honma & Tokuji Shimomura (1978) Metabolism of 1- Aminocyclopropane-1-carboxylic Acid, Agricultural and Biological Chemistry, 42:10, 1825-1831.https://doi.org/10.1080/00021369.1978.10863261
[3] Shah S, Li J, Moffatt BA, Glick BR. Isolation and characterization of ACC deaminase genes from two different plant growth-promoting rhizobacteria. https://doi.org/10.1139/w98-074
[4] Penrose DM, Glick BR. Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria. Physiol Plant. 2003 May;118(1):10-15. doi: https://doi.org/10.1034/j.1399-3054.2003.00086.x
