exaA-mCherry construct
We received the construct pCZ-exaA-lacZ from BHU. We wanted to test the activity of the exaA promoter in E. coli to check if the promoter is alcohol inducible in E. coli as well. We performed X-gal plating to check for the lacZ expression. Both the control and the test plate (having glycerol) showed blue colonies. The control plate showed blue colonies as there is the possibility of leaky expression of lacZ in pCZ.
We then decided to make a construct of exaA-mCherry in pet15b by RF Cloning. For primary PCR we used exaA in pCZ plasmid as the template to make megaprimers. A gradient PCR for the annealing temperature was set from 53-58°C.
PCR result of the megaprimer
We were expecting a band around 380bp and we got similar results in the gel. Hence we can conclude that the megaprimer formation was successful. These megaprimers and pet15b having mCherry as a reporter gene were used for secondary PCR. The annealing temperature was set at 50°C. We did DpnI digestion followed by electroporation but no colonies were seen in the plates implying that the cloning had failed.
Next, a gradient secondary PCR for the annealing temperature was set up from 51-60°C (51°C, 55°C, 58°C, 60°C) using the previously made megaprimer and the pet15b vector. After DpnI digestion and electroporation, maximum number of colonies were seen when the annealing temperatures were 58°C and 60°C. But none of the colonies grew after inoculating them in LB with antibiotic. This again implies that cloning had failed and the colonies picked up might have been satellite colonies.
We tried troubleshooting and found out that our exaA sequence forms a secondary structure. We learnt from literature that DMSO can be used in PCR to inhibit secondary structures in the DNA template or the DNA primers. So we used different concentrations of DMSO (0.5µL, 1µL, 2µL, 2.5µL) to the PCR mix and the annealing temperature was set at 58°C. Followed by DpnI digestion and electroporation, colonies were inoculated and plasmid extraction was performed. Then confirmatory PCR was done.
The band size of the clones matches with the megaprimer (about 380bp) which confirms that all the clones are positive.
pHis17-acdS construct
(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 inducing with different concentrations of IPTG 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.
pCZ-exaA-acdS construct
We isolated genomic DNA from Azospirillum lipoferum and PCR amplified the acdS gene in with with primers containing appropriate restriction sites (XbaI). A gradient PCR (for temperatures from 58°C to 66°C) was performed to amplify the acdS gene.
The pCZ-exaA vector was digested with XbaI and the digested plasmid was run on a 1% agarose gel with 1kb NEB ladder. It was confirmed that the pCZ-exaA vector is linear after restriction digestion.
This linearized vector was purified and ligated with the acdS gene and transformed into E. coli DH5α. Colony PCR was performed as a confirmatory test to check for the presence of pCZ-exaA-acdS construct. Three out of four colonies tested positive for the construct.
For thoroughness, the plasmid was extracted from these colonies using miniprep and a PCR was performed to check for the insert. One out of the three samples tested positive for the insert.
The extracted plasmid was transformed into the donor strain, E. coli S17.1 to perform the conjugation experiment (biparental mating) with Azospirillum brasilense Sp7. The plated results are shown here.
pCZ-fdhF construct
We aimed to express the hypoxia inducible E. coli promoter, fdhF in Azospirillum brasilense and see if it is active. To materialize the pCZ-fdhF construct, we started off with the restriction digestion of the pCZ plasmid with the XbaI and HindIII restriction enzymes. We confirmed the presence of linearized plasmid in 1% agarose gel electrophoresis and proceeded with gel elution for its purification. Parallely, E. coli DH5α was transformed with the pSB1C3 plasmid containing fdhF and plasmid extraction using miniprep was done.
PCR Amplification of fdhF
fdhF promoter is amplified from pSB1C3-fdhF construct with primers containing the XbaI and HindIII sites. The suitable annealing temperature for these primers were found to be 48°C after troubleshooting. Since the tracking dye is also of the same size as fdhF (a 100bp promoter), it kept masking the band and producing false negative results. Hence, the dye concentration was reduced by increasing the amount of glycerol in it. Using this modified dye, fdhF was visualized very clearly.
After troubleshooting, the following composition of the master-mix is used for fdhF.
Master mix- 20 uL
Composition:
Buffer- 2 uL
dNTP- 1 uL
Forward primer- 1 uL
Reverse primer- 1 uL
Magnesium chloride- 0.4 uL
Taq polymerase- 0.2 uL
Template- 2 uL
NFW- 12.4 uL
After PCR purification of fdhF promoter, we ligated it with the pCZ backbone and transformed it into E. coli DH5α. Colony PCR was performed with the appropriate primers to confirm the presence of the ligated pCZ-fdhF construct. On a 1.5% agarose gel we realized we could not distinguish between the amplified insert and the primer dimer band
For thoroughness, native PAGE was performed with the PCR product as an additional confirmatory test and it was observed that the fdhF promoter insert was successfully cloned.
pAK002-kanRP-acdS
We put the acdS gene extracted from Azospirillum lipoferum downstream of the constitutive promoter that codes for kanamycin resistant gene in the modified pAK002 vector. The aim of this exercise is to check the production of ACC deaminase under constitutive conditions so that it can be compared with hypoxic conditions. The acdS gene from A. lipoferum genome was PCR amplified with the appropriate restriction sites- XhoI and XmaI. Gradient PCR was performed for this purpose involving temperatures from 56°C to 66°C.
The pAK002 vector was also digested with XhoI and XmaI enzymes and ligated with the acdS insert. The ligated product was later transformed into E. coli DH5α and colony PCR was performed as a confirmatory test for the cloning. The plasmid was extracted from three positive colonies. Out of the three colonies, a plasmid PCR confirmed the clone in one of them.
References
[1] Part:BBa_K387003http://parts.igem.org/Part:BBa_K387003
[2] 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
Pellicle experiment
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. (to know more, click here). 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 Azospirillum can form a pellicle and express the exaA promoter at lower depths where hypoxic conditions are present.
To know more about the pellicle experiment, check out our Proof of Concept page!
ACC Assay
The spectrophotometric ACC deaminase assay was performed to measure the amount of α-ketobutyrate produced in the breakdown of ACC by the active ACC deaminase enzyme in E. coli. Due to time constraints we were only able to perform the assay once and the results obtained were inconclusive.
To know more about how and why the assay is performed, check out our Proof of Concept page.
Phenol Red Assay
Objective
Qualitative confirmation of ACC deaminase activity by color changing pH indicator dyes.
Background
The ACC deaminase enzyme catalyzes the conversion of ACC into ammonia and α-ketobutyrate. Phenol red is a pH indicator dye which changes color to red/pink in basic media. Therefore qualitatively when colonies containing the active acdS gene are grown on ACC containing minimal media with phenol red, they should change color.
Plan
Minimal media plates (both with and without nitrogen sources) containing phenol red (0.008%) were prepared with malate or glycerol as a carbon source and plated with ACC. Both A. brasilense pcZ:exaA as a control and A. brasilense pcZ:exaA:acdS as a test were streaked from LBA plates onto these phenol red containing plates.
Expected Results
The plate streaked with A. brasilense pcZ:exaA:acdS should have colonies which are redder in color as compared to the control plate. There would however be a basal background red color as a result of the nitrogen fixing ability of Azospirillum (however this primarily takes place under microaerobic conditions).
Results
Till the time of wiki freeze, the colonies growing on the plate were extremely small and could not be visualized clearly using the naked eye and so any change in color was not yet visible. Growth in minimal media takes a very long time for Azospirilum and it has been shown that when grown on minimal media containing glycerol as a carbon source, the doubling time is 11 hours.
References
[1] ACC Asaay: 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-x
[2] Phenol Red Assay: Patil, C., Suryawanshi, R., Koli, S., & Patil, S. (2016). Improved method for effective screening of ACC (1-aminocyclopropane-1-carboxylate) deaminase producing microorganisms. Journal of Microbiological Methods, 131, 102-104. https://doi.org/10.1016/j.mimet.2016.10.009
Growth curves
Growth curves for wild type Azospirillum brasilense sp7.
The growth curves were setup in a 96 well microplate and the instrument used was a microplate reader which measured absorbance at 600nm.
All the setups were in triplicates and the appropriate blanks were setup for each of the media composition which were then normalized with the experimental reading. Initial readings of the absorbance of all the cultures was about 0.02
Media used: Nitrogen containing minimal malate media
It can be seen from the curve that the culture has not yet reached the stationery phase and would have continued to grow had it been given more time.
Media used: Nitrogen free minimal malate media
Azospirillum brasilense is able to fix atmospheric nitrogen under microaerobic condition. As we had setup the growth curves in orbital shaking conditions, it was initially thought that the culture would be aerated and so A. brasilense would not be able to fix nitrogen and therefore the growth in nitrogen free media would be very slow initially. However, from the data obtained it is seen that after a very small initial lag phase, the culture proceeds to the exponential phase and eventually reaches the stationary phase by around 15 hours. Comparatively the culture in Nitrogen containing media has not yet reached the stationary phase in the same time.
Media used: Nitrogen containing minimal malate media with 0.25 g/L of sodium sulphite
We planned on using sodium sulphite as a hypoxia mimicking agent. Therefore, we wanted to test the toxicity of sodium sulphite on the growth of A. brasilense. From the graph we can observe that there is no significant difference in growth rates of A. brasilense in media containing and not containing sodium sulphite.
Media used: Nitrogen containing minimal malate media with 0.5 g/L of sodium sulphite.
In this growth curve a longer lag phase was observed however the final absorbance at the end of 18 hours was the highest among all the 4 media.
