iGEM 2022 — Team iiser-pune-india

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### The team notebook

---

Here is our team notebook, which documents the general timeline for various events and checkpoints associated with our project right from the beginning in January all the way till the Wiki freeze on 12th October.

---

[← Safety](https://2022.igem.wiki/iiser-pune-india/safety)
[Results →](https://2022.igem.wiki/iiser-pune-india/results)

[![](https://static.igem.wiki/teams/4392/wiki/footer/back-to-top.png "Go to top")](#top)

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* [*exaA*+*acdS*](#op1)
* [Constitutive promoter+*acdS* in *E. coli*](#op2)
* [*exaA* promoter characterisation](#op3)
* [Overexpression of EPS](#op4)
* [Metabolic modelling](#op5)
* [Climate modelling](#op6)

### *exaA*+*acdS* Construct

---

* [Cycle 1](#Cycle1_A)
* [Cycle 2](#Cycle2_A)
* [Cycle 3](#Cycle3_A)

### Cycle 1

|  |  |
| --- | --- |
| Design | To clone the *acdS* gene downstream of the *exaA* promoter in the broad host range mobilizable pCZ750 plasmid. |
| Build | Due to the design of the plasmid, it was decided to perform single restriction site digestion (XbaI) and cloning. The *acdS* gene was amplified with XbaI overhangs from the genome extracted from *A.lipoferum*. The pCZ plasmid along with the amplified *acdS* gene were digested with XbaI overnight and a ligation reaction was setup. |
| Test | The ligation product was transformed into *E. coli* DH5α and plated on tetracycline media. After overnight incubation a lot of colonies were observed and colony PCR was performed with 20 colonies, all the colonies showed negative results for the *acdS* insert |
| Learn | We realized that the cloning failed, yet there were excessive colonies on the plate as a result of self ligation of the plasmid backbone. In order to prevent this we decided to give Antarctic Phosphatase treatment to the digested plasmid backbone and then perform ligation. |

### Cycle 2

|  |  |
| --- | --- |
| Design | To give Antarctic phosphatase treatment to the digested backbone and perform cloning. |
| Build | An Antarctic Phosphatase (AP) reaction was setup as follows:  Digested plasmid: 20uL  AP buffer: 5uL  AP enzyme: 0.5uL  NFW: 24.5uL  The reaction was setup in a thermocycle with a 37°C step for 30 mins, followed by 80°C for 2 minutes to inactivate the enzyme. Ligation was performed as before. |
| Test | The ligation mixture was transformed into DH5α and plated on tetracycline media. A single colony appeared after overnight incubation. Since the efficiency of traditional cloning decreases after AP treatment, the entire procedure was repeated and three more colonies were obtained after incubation. Colony PCR was performed with the four available colonies and three colonies appeared positive for the insert in the correct orientation. |
| Learn | In order to test the orientation of a non directional cloned insert we chose forward primers from the 5' end of the promoter and reverse primers from the 3’ end of our gene. The results showed that the clone was successful |

### Cycle 3

|  |  |
| --- | --- |
| Design | To test the activity of the ACC deaminase gene in *A. brasilense* sp7 using the spectrophotometric assay designed by Honma and Shimomura. |
| Build | The *acdS* gene was cloned downstream of the *exaA* promoter in a broad host range pCZ plasmid. This engineered plasmid was then mobilized into *A. brasilense* sp7 using biparental conjugation. The expression of this gene was supposed to be verified by culturing *Azospirillum* on minimal media with various alcohols and glycerol as a carbon source (mimicking fermentative hypoxia environments). A spectrophotometric assay was planned to observe the product of the enzyme catalyzed reaction. |
| Test | The *acdS* gene was successfully cloned under the *exaA* promoter and the positive clones in *E. coli* DH5α were confirmed by colony PCR. After transforming the *E. coli* S17.1 donor strains with this engineered plasmid, conjugation was performed and transconjugated *A. brasilense* sp7 were selected by plating them on appropriate antibiotic media (tetracycline and ampicillin). Minimal media was prepaped, however it was seen that the growth of the transconjugated *Azospirillum* in minimal media was extremely slow as compared to rich LB media and so we could not proceed with further steps |
| Learn | It is possible that the metabolic load of the plasmid in minimal media is significant as even the control setup (with *A. brasilense* containing the pcZ backbone) also shows very slow growth. We might have to change our cloning backbone to some other broad host range mobilizable vector which might have lesser metabolic load in minimal media. It is also possible that we might need to optimize the composition of the minimal media to suit the growth of the trans-conjugated *Azospirillum*. |

**References:**

[1] Prigent-Combaret C, Blaha D, Pothier JF, Vial L, Poirier MA, Wisniewski-Dyé F, Moënne-Loccoz Y. Physical organization and phylogenetic analysis of acdR as leucine-responsive regulator of the 1-aminocyclopropane-1-carboxylate deaminase gene *acdS* in phytobeneficial Azospirillum lipoferum 4B and other Proteobacteria. FEMS Microbiol Ecol. 2008 Aug;65(2):202-19. <https://doi.org/10.1111/j.1574-6941.2008.00474.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] Vijay Shankar Singh, Ashutosh Prakash Dubey, Ankush Gupta, Sudhir Singh, Bhupendra Narain Singh, Anil Kumar Tripathi. Regulation of a Glycerol-Induced Quinoprotein Alcohol Dehydrogenase by σ 54 and a LuxR-Type Regulator in Azospirillum brasilense Sp7. ASM Journal. 2017, 13 June, <https://doi.org/10.1128/JB.00035-17>

### Constitutive promoter+*acdS* in *E. coli*

---

* [Cycle 1](#Cycle1_B)
* [Cycle 2](#Cycle2_B)

### Cycle 1

|  |  |
| --- | --- |
| Design | To extract the *acdS* gene from the gene synthesised pUC57-*acdS* vector using gene-specific primers we designed for *acdS*. |
| Build | *E. coli* DH5𝛼 was transformed with pUC57-*acdS*, the plasmids were extracted through Miniprep. A confirmatory agarose gel was run, and then the *acdS* gene was PCR amplified. |
| Test | The confirmatory agarose gel showed bands, but the gel ladder was not clear enough to ascertain the size of the observed bands. |
| Learn | The concentration of the gel ladder may not have been high enough, and the gel might not have run for enough time. |

### Cycle 2

|  |  |
| --- | --- |
| Design | To obtain clear bands of ~1 kB, corresponding to the *acdS* gene with a clear ladder, and no bands on the negative control lane. |
| Build | Gel run with a higher concentration of ladder, and run for a longer time at a lower voltage. |
| Test | Clear bands were observed corresponding to the *acdS* gene at 1 kB, and were correlated with a clear gel ladder, and no bands in the negative control lane. |
| Learn | The quality and concentration of the gel ladder, and the voltage and time taken to run an agarose gel, all impact the clarity of a confirmatory agarose gel electrophoresis. |

* [Cycle 1](#Cycle1_G)

### Cycle 1

|  |  |
| --- | --- |
| Design | To prepare the pHis17-*acdS* megaprimer through PCR with overlap primers, the amplified *acdS* gene, and the pHis17-mod0B plasmid as the vector backbone. Followed by PCR purification of the megaprimer. |
| Build | PCR was performed according to the protocols and calculations according to the NEB protocol for Q5® High-Fidelity 2X Master Mix. A post-PCR confirmatory agarose gel was then run. Finally, PCR purification was done. |
| Test | The post-PCR gel showed correct bands, corresponding to around 1 kB in all the 4 replicate reactions. The negative control band also showed an unexpected band. PCR purification gave the megaprimer in high yield. |
| Learn | We were not able to ascertain why the negative control band showed a band, so we performed PCR purification for the 4 replicates. |

* [Cycle 1](#Cycle1_H)

### Cycle 1

|  |  |
| --- | --- |
| Design | To prepare the pHis17-*acdS* clone plasmid through restriction free cloning using the pHis17-*acdS* megaprimer serving as both the forward and reverse primer in the PCR replication of the pHis17 backbone plasmid. |
| Build | PCR was performed according to the protocols and calculations according to the NEB protocol for Q5® High-Fidelity 2X Master Mix. A post-PCR confirmatory agarose gel was then run. |
| Test | The post-PCR gel showed us the expected band corresponding to 3.7 kb. The negative control showed no bands. |
| Learn | The gel results were inferred to have suggested that RF cloning may have succeeded. |

* [Cycle 1](#Cycle1_I)

### Cycle 1

|  |  |
| --- | --- |
| Design | To perform DpnI digestion to isolate the pHis17-*acdS* clones and confirm the presence of positive clones through agarose gel, PCR, sequencing, and SDS PAGE. |
| Build | DpnI digestion was performed, and followed up with an electroporation of *E. coli* DH5𝛼. Plasmid extraction was done with both the Test (T) and Vector Control (VC) colonies. Confirmatory PCR using *acdS*-specific primers was performed. Protein expression check followed by an SDS PAGE was done. |
| Test | Agarose gel showed presence of the expected pHis17-*acdS* (size 3.7kb) in all 5 colonies that were checked. The PCR confirmation showed positive results - the amplification of the *acdS* gene. The SDS page showed the presence of the AcdS protein at the expected band of 37kDa. |
| Learn | The RF cloning experiment seems to have succeeded. The samples were sent for sequencing for confirmation. |

**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-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. <https://doi.org/10.1034/j.1399-3054.2003.00086.x>

### *exaA* promoter characterisation

---

#### *exaA* promoter characterisation in *E. coli*

* [Cycle 1](#Cycle1_C)
* [Cycle 2](#Cycle2_C)
* [Cycle 3](#Cycle3_C)
* [Cycle 4](#Cycle4_C)

### Cycle 1

|  |  |
| --- | --- |
| Design | To test the activity of an *Azospirillum* alcohol inducible promoter *exaA* upstream of *lacZ* reporter gene in pCZ plasmid in *E. coli*. |
| Build | Minimal media plates with alcohol (glycerol) and without alcohol (control) were made and X-gal was added to the plates to study the promotor activity with the help of lacZ expression. |
| Test | Blue colonies were seen in plates with and without alcohol. |
| Learn | After going through some literature on pCZ plasmid, we found that leaky expression of *lacZ* is seen in pCZ plasmid. We decided to change our vector in order to obtain conclusive results. |

### Cycle 2

|  |  |
| --- | --- |
| Design | To clone *exaA* promotor upstream of fluorescent reporter mCherry in pet15b vector via RF Cloning. |
| Build | *exaA* promoter in pcZ plasmid was used as a template to make megaprimer for RF Cloning. Megaprimer and the recipient plasmid pet15b were used in the secondary PCR. The annealing temperature was set to 50°C. PCR products were digested by Dpn 1 enzyme. Digested products were introduced in NEB electrocompetent *E. coli* by electroporation. |
| Test | Colonies were not seen in the plates, implying that cloning had failed. |
| Learn | The annealing temperature might have been too low. The secondary PCR should be performed with a gradient of temperatures to identify the optimum annealing temperature. |

### Cycle 3

|  |  |
| --- | --- |
| Design | To setup a gradient secondary PCR to find the optimal annealing temperature. |
| Build | While performing the secondary PCR, megaprimer made during the first cyle were used. A gradient from 51°C-60°C was set up for the secondary PCR. Electroporation with digested PCR product was done. |
| Test | Maximum colonies were seen when the annealing temperature was 58°C and 60°C. After inoculation, the culture did not grow well. |
| Learn | For troubleshooting we referred to literature describing the promotor and we found that *exaA* promoter forms a secondary structure which might interfere with RF Cloning process |

### Cycle 4

|  |  |
| --- | --- |
| Design | To perform secondary PCR with DMSO to inhibit secondary structures in *exaA* promoter sequence |
| Build | Different concentrations of DMSO were added to the PCR mix. The annealing temperature was set to 58°C; Secondary PCR was performed. |
| Test | After the transformation of digested PCR products into NEB electrocompetent cells, the culture after inoculation showed growth. Confirmatory PCR revealed that our clones were positive. |
| Learn | Two of our samples have been given for sequencing for final confirmation, but we haven’t received the sequencing results yet. After the final confirmation, we plan on characterising the activity of the promotor by preparing minimal media and adding different alcohols in varying concentrations and observing the fluorescence readout. |

**References:**

[1] Vijay Shankar Singh, Ashutosh Prakash Dubey, Ankush Gupta, Sudhir Singh, Bhupendra Narain Singh, Anil Kumar Tripathi. Regulation of a Glycerol-Induced Quinoprotein Alcohol Dehydrogenase by σ 54 and a LuxR-Type Regulator in Azospirillum brasilense Sp7. ASM Journal. 2017, 13 June, <https://doi.org/10.1128/JB.00035-17>

[2] Nelms, B., Labosky, P. A predicted hairpin cluster correlates with barriers to PCR, sequencing and possibly BAC recombineering. Sci Rep 1, 106 (2011). <https://doi.org/10.1038/srep00106>

[3] Eric M. Ransom, Craig D. Ellermeier, David S. Weiss. Use of mCherry Red Fluorescent Protein for Studies of Protein Localization and Gene Expression in Clostridium difficile. ASM Journal. 2015, 10 February.[https://doi.org/10.1128/AEM.03446-14](https://doi.org/10.1128/AEM.03446-14%20)

#### *exaA* promoter characterisation in *Azospirillum*

* [Cycle 1](#Cycle1_J)

### Cycle 1

|  |  |
| --- | --- |
| Design | To test the induction of the *exaA* promoter in *Azospirillum brasilense*  sp7 using the *lacZ* reporter gene under microaerobic condition. |
| Build | pCZ-*exaA*:*lacZ* construct was mobilized into *A. brasilense*. A pellicle experiment was setup, in both nitrogen free as well as nitrogen containing semi solid media to allow *Azospirillum* to migrate as a band to a microaerobic environment and allow gene expression. X-gal was used as a chromogenic substrate to observe the test band compared to the band generated by the control (pCZ-*lacZ* vector). |
| Test | The transconjugated *Azospirillum* could grow in the appropriate antibiotic media confirming that the plasmid had been mobilized. In the nitrogen free media, it was visually observed that the blue band generated by pCZ-*exaA*:*lacZ* appeared darker and thicker than the control setup of the pCZ-*lacZ* backbone without the *exaA* promoter. However, the control setup also showed a blue band and quantitative estimates could not be obtained. |
| Learn | We need to perform Miller’s assay to quantify the activity of promoter expression and get quantitative data for the test vs the control. We also realized that the pCZ-*lacZ* backbone shows background basal expression of beta galactosidase and this interferes with the analysis of the result. It would be better to subclone this entire construct into a new broad range mobilizable vector which has limited background expression. |

### Overexpression of EPS

---

* [Cycle 1](#Cycle1_D)

### Cycle 1

|  |  |
| --- | --- |
| Design | To overexpress UDDP-glucose–epimerases (*ExoB*and *ExoB2*) and Phosphomannomutase (*ExoC*) in pCZ750 plasmid. |
| Build | We plan on cloning *ExoB*, *ExoB2* and *ExoC* downstream of alcohol inducible *exaA* promotor by traditional cloning method. |
| Test | We plan on isolating and quantifying EPS produced after cloning the desired genes by phenol sulphuric acid method. To induce the activity of the genes producing EPS we will prepare minimal media plates with glycerol and Tetracycline for culturing the clones. |
| Learn | We expect that we will successfully over-express EPS and study the nature of different EPS produced by the respective genes. |

**References:**

[1] Kaur, T., & Ghosh, M. (2015). Characterization and upregulation of bifunctional phosphoglucomutase/phosphomannomutase enzyme in an exobiopolymer overproducing strain of Acinetobacter haemolyticus. Microbiological Research, 181, 8-14.<https://doi.org/10.1016/j.micres.2015.08.003>

### Metabolic Modelling

---

* [Cycle 1](#Cycle1_E)
* [Cycle 2](#Cycle2_E)
* [Cycle 3](#Cycle3_E)
* [Cycle 4](#Cycle4_E)

### Cycle 1

|  |  |
| --- | --- |
| Design | The Genome Scale Model (GSM) of our chassis- *Azospirillum brasilense* Sp7 was required to perform Flux Balance Analysis and optimisation, so that the confidence in the predictions and their accuracy would be high. |
| Build | Since no such GSM was available, we made our own GSM using automation algorithms provided by the ModelSEED platform. |
| Test | While setting constraints on the model, we realized the reaction and metabolite names were set arbitrarily by the algorithm, and un-curated model such as this would have a lot of gaps in its reaction pathways. |
| Learn | From the advice of Dr. Anu and Dr. Karthik, we understood that manually curating this model would be extremely infeasible since the reaction and metabolite names would have to be individually searched for and input.  Dr. Anu then suggested that we use the GSM for *Azospirillum lipoferum* B510, which she provided us with from Biomodel.org. Since it was from a public database and would probably be well curated. |

### Cycle 2

|  |  |
| --- | --- |
| Design | We began working on the A.B510 GSM by first making sure that the reaction and metabolite names have the standard nomenclature. Since it looked in order, we shifted our entire design to be based on the A.B510 GSM. |
| Build | We set constraints of the Exchange reactions as required- input reactions would have a lower bound of -1000 and upper bound of 0 and output reactions would have lower bound of 0 and upper bound of 1000. |
| Test | To check for internal inconsistencies, we then ran FBA with 0 carbon source as input as a sanity check. The expectation was to get 0 growth rates. We also ran FBA with some positive values for the carbon sources, where the expectation was to get some non-zero value as the growth rate. |
| Learn | We got 0 growth rates in all the situations, implying that there was some issues with the model. The first hypothesis was that there are gaps in this model too. |

### Cycle 3

|  |  |
| --- | --- |
| Design | To find the gaps, we ran the GSM findGaps.m function and worked through the FastGapFill tutorial provided by the Cobra Toolbox developers on their Github. |
| Build | We found 277 root gaps, which lead to 1569 reactions being blocked downstream. Since this number was too big to ignore, we tried running the fastGapFill function. |
| Test | After a lot of troubleshooting with the code, we found that the Cobratoolbox developers had an error in their code on Github, which was presumably giving us errors at our end. We emailed them, and this issue was resolved at their end in a few days.  We then tried gap-filling again, but there were 0 added reactions to our model, which meant that the function was filling gaps without having to add any new reactions. |
| Learn | After some more troubleshooting, we learned that the function was changing the reaction bounds of exchange reactions to allow both-way flow- into the bacteria as well as out of it. This did not make biological sense for excretory metabolites. We then had no option but to manually look into the dead-ends to see why this issue was occurring. After doing about 15 dead ends, it was evident that the manual curation of the model would again be infeasible. We also learned that the GSM had been made using automation software by the authors too, and we had come full circle back to square one! |

### Cycle 4

|  |  |
| --- | --- |
| Design | We decided to look for a fully curated GSM of the closest related bacteria to *Azospirillum*. Since *Pseuodomonas* is closely related to *Azospirillum*, and it has well established GSMs, we decided to use this GSM to make order-of-magnitude approximations for our system. We could also test whether our logic and workflow is valid and then have the code and its analysis ready for when an *Azospirillum* GSM is available to us. |
| Build | We made the GSM complete by adding the reactions specific to our system and the metabolites needed for the same. |
| Test | From there, we tested the flux through biomass with 0 input carbon sources and free carbon sources, to see if biomass is being produced without having any input, and checking that we have positive flux with free carbon input. |
| Learn | We then ran simulations to extract the maximum rate at which our system can degrade ACC, and the dependencies of ACC and oxygen to the growth rates of the bacteria. |

**References:**

[1] Lewis, Leah & Perisin, Matthew & Tobias, Alex. (2020). Metabolic modeling of Pseudomonas putida to understand and improve the breakdown of plastic waste. <http://dx.doi.org/10.13140/RG.2.2.25853.28643>

[2] Model iJN1463, bigg models database<http://bigg.ucsd.edu/models/iJN1463>

### Climate Modelling

---

* [Cycle 1](#Cycle1_F)
* [Cycle 2](#Cycle2_F)
* [Cycle 3](#Cycle3_F)
* [Cycle 4](#Cycle4_F)

### Cycle 1

|  |  |
| --- | --- |
| Design | To find the water covered regions from Sentinel-2 optical data using masking. |
| Build | Masking algorithm was coded in python. Sentinel-2 optical data for a district in Punjab was downloaded. |
| Test | Masking was performed in the downloaded Sentinel-2 data. The masking was found to be ineffective when correlated with real-life data. |
| Learn | We realised that masking is not a good algorithm to work with in this case. |

### Cycle 2

|  |  |
| --- | --- |
| Design | To find the water covered regions from Sentinel-2 optical data using image segmentation. |
| Build | K-means clustering algorithm was coded in python. Sentinel-2 optical data for a district in Punjab was downloaded. |
| Test | Clusters were found using k-means clustering and then the water pixels were labelled. These water pixels were different from the real-life data and hence the model was ineffective. |
| Learn | We realised that image segmentation using k-means is not a good algorithm to work with in this case. |

### Cycle 3

|  |  |
| --- | --- |
| Design | To find the water covered regions from Sentinel-2 optical data using image segmentation using R-CNN. |
| Build | R-CNN was coded in python. Sentinel-2 optical data for a district in Punjab was downloaded. |
| Test | Clusters were found using R-CNN and then the water pixels were labelled. These water pixels were different from the real-life data and hence the model was ineffective. |
| Learn | We realised that image segmentation using R-CNN is not a good algorithm to work with in this case. The training set was probably ineffective. |

### Cycle 4

|  |  |
| --- | --- |
| Design | To find the waterlogged regions using QGIS and SNAP. |
| Build | NDWI,NDVI,NDMI and MNDWI ratios were calculated. Sentinel-2 bands data for a district in Punjab was downloaded. Cutoffs for all these ratios were found for waterlogging areas. |
| Test | All the ratio maps were calculated and the maps were combined together to find waterlogging hotspots. |
| Learn | We found waterlogged areas for the district. We can extend this algorithm to the entire country. |

**References:**

[1] Arnous, Mohamed O., and David R. Green. “Monitoring and Assessing Waterlogged and Salt-Affected Areas in the Eastern Nile Delta Region, Egypt, Using Remotely Sensed Multi-Temporal Data and GIS.” Journal of Coastal Conservation 19, no. 3 (2015): 369–91.<http://dx.doi.org/10.1007/s11852-015-0397-5>

[2] Kaushik, S., Dhote, P.R., Thakur, P.K. et al. An integrated approach for identification of waterlogged areas using RS and GIS technique and groundwater modelling. Sustain. Water Resour. Manag. 5, 1887–1901 (2019).[http://dx.doi.org/10.1007%2Fs40899-019-00342-1](http://dx.doi.org/10.1007/s40899-019-00342-1)

[← Contribution](https://2022.igem.wiki/iiser-pune-india/contribution)
[Experiments →](https://2022.igem.wiki/iiser-pune-india/experiments)

[![](https://static.igem.wiki/teams/4392/wiki/footer/back-to-top.png "Go to top")](#top)

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* [RF Cloning](#e1)
* [α-ketobutyrate standard curves](#e2)
* [Expression of *acdS* gene](#e3)
* [ACC deaminase assay](#e4)

* [Genomic Extraction and Amplification](#a1)
* [*Azospirillum* specific PCRs](#a2)
* [Traditional cloning](#a3)
* [Competent cell preparation](#a4)
* [Conjugation](#a5)
* [Pellicle](#a6)
* [EPS Quantification](#a7)

* **Overview**
* ***E. coli***
* ***Azospirillum***

### Overview

---

Our wetlab consisted of two parts: experiments using *Azospirillum brasilense* sp7 and *E. coli*. *Azospirillum brasilense* is our chassis organism but since we are the first iGEM team to work with it, we also decided to simultaneously work with *E. coli* as a model system. We decided to work in Prof. Anil Kumar Tripathi’s lab at Banaras Hindu University as he is one of the leading scientists working on *Azospirillum*.

To begin with we aimed to characterize the activity of the alcohol inducible promoter, known as the *exaA* promoter in hypoxic environments. In order to do so we planned on performing a pellicle experiment assay which would enable *Azospirillum* to migrate to a microaerobic environment which would have allowed us to ascertain the activity of the *exaA* promoter.
We also planned on working with the *fdhF* promoter, which is a well characterized hypoxia promoter in *E. coli*. To do so we planned on cloning the *fdhF* promoter upstream of the *lacZ* gene in the pcZ backbone by traditional cloning.

As a proof of concept we planned on cloning the *acdS* gene downstream of the *exaA* promoter in the pCZ vector and mobilizing this engineered plasmid into *A. brasilense* by biparental mating. As a qualitative assay we wanted to monitor the growth curves of the transconjugated *Azospirillum* in order to determine the metabolic load/benefit of our engineered plasmid.

In order to have a proper control to check hypoxic expression vs regular expression we also planned on cloning the *acdS* gene downstream to a constitutive promoter in the pAK002 plasmid and use this as a comparative study for all of our experiments involving *acdS* expression.

To characterize the activity of the ACC deaminase gene we planned on performing a spectrophotometric assay to detect the product formed as a result of the enzyme catalyzed reaction.

As we were working with two organisms we decided to conduct a cross-species promoter study in order to characterize a new probable alcohol inducible/hypoxia promoter in *E. coli*. To characterise *exaA* in *E. coli* we planned on cloning it upstream of the *mCherry* reporter gene by using RF cloning. Since it might have been difficult to perform the ACC assay in *Azospirillum* we planned on performing it in *E. coli* and then customising the protocol for *E. coli*. To do the same we decided to clone the *acdS* gene downstream of T7 overexpression promoter.

To find the detailed protocol for all these procedures refer the following tabs.

[← Engineering](https://2022.igem.wiki/iiser-pune-india/engineering)

### RF Cloning

---

1. First step of RF cloning involves designing primers for the primary PCR. Primers are made by taking two parts into consideration :
   1) The sequence flanking the region where target gene has to be introduced in the recipient vector
   2) 5’ and 3‘ ends of the target gene.
2. Primary PCR :
   This PCR product, called the megaprimer, will have the target gene flanked by regions complementary to the regions flanking the site of insertion in the target vector. The plasmid with the target gene or linearised target gene will be used as the template of this PCR.
3. Secondary PCR :
   Purified PCR product from the primary PCR will be used for the secondary PCR. The template vector will be the recipient vector and during amplification, the target gene will be inserted into the recipient vector.
4. After the secondary PCR, the product is digested with Dpn 1 to degrade methylated parent vectors. The digested product is introduced in E.coli by electroporation.

Primary PCR protocol :

1. Initial denaturation: Run for 2 min at 95°C
2. Repeat 30 times:
a) Denaturation: 30s at 95°C
b) Annealing: 30s at the desired annealing temperature
c) Extension: 1 min/1 kb template at 72°C
Note : The rate of extension depends on the polymerase being used in the PCR mix. The rate for Pfu polymerase has been mentioned.
3. Final extension: Run for 10 min at 72°C
4. Run PCR products on an agarose gel and purify it using a PCR purification kit.
5. Dilute the final concentration to 100ng/μL

Secondary PCR protocol :1. Initial Denaturation: Run for 5 min at 95°C
2. Repeat 30 times:

a)Denaturation: 30s at 95°C
b)Annealing: 30s at the desired annealing temperature
c)Extension: 1 min + 1 min/1 kb template at 72°C
Note: The rate of extension depends on the polymerase being used in the PCR mix. The rate for Pfu polymerase has been mentioned.
3. Final extension : Run again for 10 min at 72°C.
4. Run PCR products on an agarose gel.
5. Digest the PCR products using DpnI enzyme at 37℃ for 3 hrs.
6. Transform the product into electrocompetent *E. coli* by electroporation.

### α-ketobutyrate standard curves

---

1. Make 2M Tris HCl pH 8.5
2. Volume to be made - 200mL
   - Procedure
   a) Weigh 48.456g of Tris base and add it to 140mL of milliQ
   b) Adjust pH to 8.5 by adding conc HCl and let it cool to room temp and check pH again - readjust if changed
   c) Make volume to 200mL and autoclave

3. Make 0.1M Tris HCl pH 8.5

1. Volume to be made - 100 mL
2. Procedure
a)Take 5mL 2M Tris HCl and add to 95mL of autoclaved milliQ

4. Make 100mM stock of α-KB(AKB)

1. Volume to be made - 25mL, in a falcon
2. Procedure
a) Measure 0.255g of AKB and add it to 25mL of 0.1M Tris HCl

5. Make 10mM working solution of AKB

1. This is to be done just before working only
2. Volume to be made - 5mL
3. Procedure
a) Take 500uL of 100mM AKB and add to 4.5mL of 0.1M Tris HCl

6. Make aliquots of AKB of different concentrations

1. Volume to be made - each aliquot is finally 200uL
2. Label them as
a) 0.5mM AKB - add 10uL of 10mM AKB in 190uL of 0.1M Tris HCl
b) 1.0mM AKB - add 20uL of 10mM AKB in 180uL of 0.1M Tris HCl
c) 1.5mM AKB - add 30uL of 10mM AKB in 170uL of 0.1M Tris HCl
d) 2.0mM AKB - add 40uL of 10mM AKB in 160uL of 0.1M Tris HCl
e) 2.5mM AKB - add 50uL of 10mM AKB in 150uL of 0.1M Tris HCl
f) 3.0mM AKB - add 60uL of 10mM AKB in 140uL of 0.1M Tris HCl
g) 3.5mM AKB - add 70uL of 10mM AKB in 130uL of 0.1M Tris HCl
h) 4.0mM AKB - add 80uL of 10mM AKB in 120uL of 0.1M Tris HCl
i) 4.5mM AKB - add 90uL of 10mM AKB in 110uL of 0.1M Tris HCl
j) 5.0mM AKB - add 100uL of 10mM AKB in 100uL of 0.1M Tris HCl

### Expression of *acdS* gene

---

1. Transform *E.coli* BL21DE3 with pHis17-*acdS*
2. The next morning, pick up a patch from the plate and inoculate it in 10mL LB + Amp
3. Incubate it at 37°C, 180rpm until turbidity is observed
4. Prepare 4 test tubes, labelled as:
a) Uninduced
b) 0.25mM IPTG
c) 0.50mM IPTG
d) 1.00mM IPTG
5. Add the following to the tubes:
a) 10mL LB+Amp
b) 100uL of primary culture
6. Incubate at 37°C, 180rpm until the OD is between 0.4-0.8
7. Once the OD is in range, add the following to the tubes:
a) Uninduced - 0uL of 1M IPTG
b) 0.25mM IPTG - 2.3uL of 1M IPTG
c) 0.5mM IPTG - 4.6uL of 1M IPTG
d) 1mM IPTG - 9.1uL of 1M IPTG
8. Induce for 3 hours at 37°C
9. Take 3mL of each culture, pellet it down, flash freeze it, and store it at -80°C.

### ACC deaminase assay

---

1. Transform *E. coli* BL21(DE3) with pHis17-*acdS*
2. Do primary inoculation in 10mL LB + Amp media, 37°C for 18 hours
3. Harvest cells by centrifugation, at 6000g for 10 mins in a 4° incubator
4. Throw out the supernatant
5. Wash twice with 0.1M Tris-HCl (pH 7.5) (Resuspend, pellet down, throw supernatant)
6. For secondary inoculation, do it in duplicate, 10mL M9 minimal media with 0.5mM IPTG (5uL of 1M IPTG), for 18 hours
7. The cells are then pelleted and resuspended in 0.1M Tris-HCl (pH 8.5)
8. The cells are then lysed by sonication
9. In a 1.5mL MCT, 200uL of sonicated cells are added
10. 20uL of 0.5M ACC is added
11. The suspension is vortexed, then incubated at 30°C for 15 mins
12. 1mL of 0.56M HCl is added
13. The mixture is vortexed, then centrifuged at 25°, 16000g, 5 mins
14. Into a glass test tube, add 1mL of supernatant and 800uL of 0.56M HCl, vortex
15. Then 300uL of 0.2% 2,4-DNP in 2M HCl is added to the tube
16. The contents are vortexed and incubated at 30°C for 30 mins
17. 2mL of 2N NaOH is added and mixed
18. The absorbance of the mixture is measured at 540nm

### 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-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. Can J Microbiol. 1998 Sep;44(9):833-43. PMID: 9851025<http://dx.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. <https://doi.org/10.1034/j.1399-3054.2003.00086.x>

### Genomic Extraction and Amplification

---

1. Harvest cells from 2mL of bacterial culture by centrifuging at 14,000 rpm for 2 mins. Collect the pellet and discard the supernatant.
2. Resuspend cells in 567uL of TE buffer.
3. Add 30uL of 10% SDS and 3uL of proteinase K to the solution and invert mix it.
4. Incubate at 37°C for 40-60 mins till the solution is colorless.
5. Add 100uL of 5M NaCl, mix by vortexing and add 80uL of pre-warm CTAB solution.
6. Mix it by vortexing and incubate at 65°C for 10 mins in a water bath.
7. Add equal volume of PCI (Phenyl Chloroform Isoamyl alcohol). Mix it by vortexing for about 2 mins.
8. Centrifuge at 14,000 rpm for 10 mins. Separate the upper aqueous layer in another eppendorf (take around 600uL) and add 0.6 times volume (usually 360uL) of chilled isopropanol.
9. Centrifuge at 14,000 rpm for 10 mins at room temperature.
10. Discard supernatant and wash pellets with 1mL of 70% ethanol.
11. Add 0.7mL of 100% ethanol to 0.3mL of water in a 1.5mL eppendorf tube.
12. Centrifuge at 14,000 rpm for 5 mins at room temperature.
13. Discard supernatant and air dry the pellet in 37°C for 5 mins.
14. Add 50uL of NFW, vortex properly and add 1uL of RNAse.
15. Incubate at 37°C for 1 hour. Store it in 4°C if required.

### *Azospirillum* specific PCRs

---

The following modified master mix is used for PCR involving the *Azospirillum* genome (for both *A. brasilense* and *A. lipoferum*).

1. Master Mix composition:
a) Taq polymerase - 1uL
b) Taq buffer (10X)- 10uL
c) Primers (forward and reverse)- 5uL each
d) dNTP - 5uL
e) DMSO - 5uL
f) NFW- add so that the total volume is 100uL
g) Template- 0.5uL for genomic DNA and 2uL of plasmid DNA
2. After adding all these components in a 1.5mL eppendorf tube, pipette mix properly and distribute the master-mix in PCR tubes kept in ice.
3. Short spin the PCR tubes and set the tubes in PCR machine.
   DMSO is added in the PCR master-mix because Azospirillum genome is GC rich.

PCR cycle:
a) Initial denaturation: 95°C for 5 mins
b) Denaturation: 95°C for 1 min
c) Annealing: Optimized temperature determined by gradient PCR for 1 minute
d) Extension: 72°C for 1 min (for 1 kb gene or less), 2 min (for 2 kb gene) and 3 min (for 3 kb gene)
e) Storage: 4°C for as long as required

### Traditional Cloning

---

1. Restriction digestion:
   For a 50uL double digestion, add the below constituents in the following order:
   a) NFW - 23.5uL
   b) Plasmid - 20uL
   c) Buffer - 5uL
   d) Enzyme 1 - 0.75uL
   e) Enzyme 2 - 0.75uL
   This is followed by overnight incubation at the appropriate temperature depending on the restriction enzyme used.
2. Antarctic phosphatase reaction:
   Add the below constituents in the following order:
   a) Digested plasmid- 50uL
   b) NFW- 24.5uL
   c) Antarctic phosphatase buffer 10x - 5uL
   d) Antarctic phosphatase enzyme- 0.5uL
   Total- 50uL
   The reaction is setup in a thermocycle with a 37°C step for 30 mins, followed by 80°C for 2 minutes to inactivate the enzyme.
3. Ligation of an insert in the vector of interest:
   Add the below constituents in the following order into a 1.5mL Eppendorf:
   a) NFW - 5uL
   b) Digested plasmid - 8uL
   c) Insert - 4uL
   d) Buffer - 2uL
   e) Enzyme - 1uL
   f) ATP - 0.5 uL
   Place the Eppendorf in a PCR machine for 10 minutes at 22°C, followed by 3 hours at 16°C.
4. The ligation product is then transformed into *E. coli* DH5α and plated on the appropriate antibiotic media.
5. Colony PCR:
   Preparation of 1mL Titron X:
   a) Add 2-3uL of 100x Titron X to 1mL of autoclaved ddH2O.
   b) Add 20uL of this mix in the respective 1.5mL Eppendorf tubes.
   c) Pick up individual colonies from the plate and mix it properly with titron X and then short vortex the tubes.
   d) Insert the Eppendorf tubes in boiling water for 10 mins and short vortex again.
   e) Centrifuge at 14000 rpm for 5 mins. This is now the template for the PCR reaction.
   f) Template should be added in the master mix such that template is 0.1 times the volume of master mix.

### Competent cell preparation

---

1. Setup a primary 5mL primary culture and allow overnight incubation.
2. Inoculate secondary culture from primary culture. For *E. coli*, 0.5% - 1% volume from the primary culture should be inoculated.
3. Harvest 50mL of cells when the OD600 of the culture is around 0.6.
4. Centrifuge the culture at 4000rpm at 4°C for 10 mins.
5. Discard the supernatant and resuspend the cells in chilled 10mL 100mM CaCl2 and incubate it in ice for 30 mins.
6. Centrifuge at 4000rpm for 10mins at 4°C. Discard the supernatant.
7. Dissolve the cells in 25% (250mL) glycerol and 75% (750mL) 100 mM CaCl2.
8. Pour it in 1.5mL eppendorf tubes and store it in -80°C for further use.

### Conjugation: Biparental Mating

---

1. Plating the donor and the recipient together:
a) Day 1: Primary inoculation of *E. coli* S17.1 and *Azospirillum brasilense* Sp7 in LB with antibiotics.
b) Day 2: Secondary culture of S17.1 (50-100uL in 5 mL LB) and Sp7 (250uL in 5 mL LB) with appropriate antibiotics.
c) Monitor OD till it reaches 0.6 - 0.8.
d) Collect 1:3 ratio of *E. coli* S17.1: *Azospirillum brasilense* and mix.
e) Centrifuge at 4000 rpm for 3 mins.
f) Discard the supernatant and resuspend in 1mL of 0.85% saline solution.
g) Add 100uL of above mixture at the center of a blank LBA plate dropwise.
9) Keep the plate without parafilm in a humid chamber (made by layering tissue papers and cotton inside a tip box and wetting it) at 30°C.
10) Check 2-3 days later for growth and then move onto the spreading step.

- Spreading and dilution:
a) Take 1mL of 0.85% saline solution in 1.5mL eppendorf.
b) Using a 1mL tip, pickup the central portion of *Azospirillum brasilense* Sp7 growth. It is observed that there is growth of *Azospirillum* in the center and surrounding its periphery, with a distinct margin, is *E. coli* growth.
c) Swirl and add to the 1mL saline solution in the eppendorf. Keep on scraping and swirling and mixing till nearly all *Azospirillum* has been removed from tip. Make sure not to touch *E. coli*.
d) Pipette mix the saline solution and *Azospirillum* together using 1 mL tip. As the mixture will have some solid suspended, allow it to sediment by keeping it vertical for some time.
e) Meanwhile, take 4 eppendorf tubes for dilution and label them (-1,-2,-3 and -4).
f) Make 5 plates having Ampicillin (for *Azospirillum*) and the corresponding antibiotic of the plasmid to be mobilized. Label the 5 plates as Sp7:plasmid from 0 to -4.
g) Add 900uL saline solution to the -1 tube and then add 100uL of supernatant (having *Azospirillum*) formed from the previous tube. Continue doing the same for the subsequent tubes (serial dilution).
h) For spreading, take 100uL from each medium and drop it at various locations on separate plates and spread thoroughly.

### Pellicle

---

Step 1: Preparing primary culture
1. Take 5mL of complete minimal malate solution (NFB+) in test tubes.
2. Add appropriate concentrations of antibiotics.
3. Scrape out single colonies from the respective *Azospirillum* plate using a pipette.
4. Discard the pipette tip inside the test tubes.
5. Keep the test tubes in an incubator at 30°C.
6. Wait until the OD becomes 1.

Step 2: Preparing secondary culture
1. After the OD becomes 1, inoculate secondary culture.
2. Add 5mL of minimal malate media in a test tube and with the appropriate antibiotics.
3. Add 200uL of primary culture to this test tube.
4. Keep it in an incubator for around 8 hours.

Step 3: NFB- broth preparation
Add the following solutions in a 10mL falcon:
1. 500uL of phosphate solution
2. 500uL of malate solution
3. 500uL of N2 free salt solution
4. Add autoclaved ddH2O until the volume is 10mL.

Step 4: Inoculum preparation
1. The secondary culture would have an OD of 0.8-0.9.
2. Collect 1 OD cells from the secondary culture.
3. Volume that is supposed to be collected = Desired OD/Actual OD.
4. Centrifuge the cells at 4000 rpm for 3 mins.
5. Discard the supernatant and resuspend the pellet in 1mL NFB- broth by tapping.

Step 5: 0.2% Agar (semi-solid media) preparation
1. Dissolve 0.2g Agar in 85mL ddH2O and autoclave it.
2. Add 5mL of each solution: phosphate, N2 free salt and malate solutions.
3. To 30mL solution of this, add 30uL of ampicillin.
Note: *Azospirillum brasilense* has ampicillin resistant beta lactamase genes in its genome.

Step 6: Inoculum setup
1. In 5mL of semisolid media that has still not solidified add the required antibiotics (appropriate with respect to the plasmid inserted in the chassis) and relevant reagents in appropriate amounts.
2. Pipette 50uL of cell mixture and very slowly and stably release it in the layer just below the top.
3. Once inoculated, make sure that the test tube undergoes minimum shaking or movement and keep it at 30°C (for growth) or room temperature (if only movement has to be observed) in an extremely stable place.
4. Check pellicle after 24-48 hours.

### EPS Quantification

---

1. Centrifuge biofilm samples (15mL of A. brasilense culture) at 15,000g for 20 min.
2. Resuspend pellets in 30mL of cold 0.2M sulphuric acid solution.
3. Break biofilm matric with a glass hand homogeniser tube and pestle.
4. Stir cell suspension at 4°C for 3 hours before centrifuging at 15,000g for 20 min.
Note: this is now known as “EPS solution”
5. Chemical composition analysis:
a. Precipitate EPS by adding 3 volumes of 100% cold ethanol and incubate on ice for 2 hours.
b. Precipitate is centrifuged at 17,500g for 2 minutes at 4°C and dried at 50°C overnight.
c. Perform FTiR spectroscopy.
6. Total carbohydrate content:
a. 50µL of EPS solution is mixed with 125µL of concentrated sulfuric acid.
b. 25µL of 10% phenol is mixed in and mixtures is incubated at 95°C water bath for 5 minutes.
c. Allow mixture to cool then transfer to a 96-well plate.
d. Measure absorbance at 490nm with a spectrophotometric plate reader.

[Measurement →](https://2022.igem.wiki/iiser-pune-india/measurement)

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

* [*exaA*-*mCherry* construct](#u1)
* [pHis17-*acdS* construct](#u2)
* [pCZ-*exaA-acdS* construct](#u3)
* [pCZ-*fdhF* construct](#u4)
* [pAK002-*kanRP-acdS* construct](#u5)
* [References](#u6)

* [Pellicle Experiment](#d1)

* [ACC assay](#i1)
* [Phenol Red Assay](#i2)
* [References](#i3)

* [In NFB(+) media](#e1)
* [In NFB(-) media](#e2)
* [In 0.25g/L Sodium sulphite](#e3)
* [In 0.5g/L Sodium sulphite](#e4)

* **Cloning**
* **Promoter Assays**
* **ACC Assays**
* **Growth Curves**

### *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.

![](https://static.igem.wiki/teams/4392/wiki/results/exaa-mcherry/xgalcontrol.jpg)
*Plates containing *E. coli* with pCZ-*exaA* and X-gal without glycerol*

![](https://static.igem.wiki/teams/4392/wiki/results/exaa-mcherry/xgalglycerol.jpg)
*Plates containing *E. coli* with pCZ-exaA, X-gal and glycerol*

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

 ![](https://static.igem.wiki/teams/4392/wiki/results/exaa-mcherry/megaprimer.png)
 *Lane 1: 1kb ladder, Lane 2: Negative Control(No template), Lane 3: Sample1(53°C), Lane 4: Sample 2(54°C), Lane 5: Sample 3(55°C), Lane 6: Sample 4(56°C), Lane 7: Empty, Lane 8: Sample 5(58°C)*

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.

![](https://static.igem.wiki/teams/4392/wiki/results/exaa-mcherry/exaaconfirm.png)
*Lane 1: 1kb ladder, Lane 2: Positive Control(PCR for Megaprimer), Lane 3: Sample 1(0.5µL DMSO), Lane 4: Sample 2(1µL DMSO), Lane 5: Sample 3(2µL DMSO), Lane 6: Sample 4(2µL DMSO), Lane 7: Sample 5(2.5µL DMSO), Lane 8: Sample 6(2.5µL DMSO)*

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*

![](https://static.igem.wiki/teams/4392/wiki/proof-of-concept/gel-1.png)

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

![](https://static.igem.wiki/teams/4392/wiki/proof-of-concept/gel-2.png)

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

![](https://static.igem.wiki/teams/4392/wiki/proof-of-concept/gel-3.png)

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

![](https://static.igem.wiki/teams/4392/wiki/proof-of-concept/plates-4.png)

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*

![](https://static.igem.wiki/teams/4392/wiki/proof-of-concept/gel-4.png)

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*

![](https://static.igem.wiki/teams/4392/wiki/proof-of-concept/sds-page.png)

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.

![](https://static.igem.wiki/teams/4392/wiki/results/clones/pcr-amplification-acds.jpg)
 *The gradient PCR with amplified acdS*

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.

![](https://static.igem.wiki/teams/4392/wiki/results/clones/restriction-digestion-pcz-exaa.jpg)
 *1% agarose gel with linearized plasmid*

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.

 ![](https://static.igem.wiki/teams/4392/wiki/results/clones/colony-pcr-pcz-exaa-acds.jpg)
 *Colony PCR results in 1% agarose gel*

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.

 ![](https://static.igem.wiki/teams/4392/wiki/results/clones/plasmid-extraction-pcz-exaa-acds.jpg)
 *The extracted plasmid in 1% agarose gel*

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.

 ![](https://static.igem.wiki/teams/4392/wiki/results/0dilution-igem.jpg)
*0th dilution plate: spreading of *Azospirillum brasilense* sp7 having the pCZ-exaA-*acdS* construct onto LBA plate ( with Ampicillin, the selective marker for Azospirillum and tetracycline, the selective marker for pCZ plasmid present in the media)*

![](https://static.igem.wiki/teams/4392/wiki/results/1dilution-igem.jpg)
 *-1 dilution plate: spreading of *Azospirillum brasilense* sp7 having the pCZ-exaA-*acdS* construct onto LBA plate ( with Ampicillin, the selective marker for Azospirillum and tetracycline, the selective marker for pCZ plasmid present in the media)*

![](https://static.igem.wiki/teams/4392/wiki/results/2dilution-igem.jpg)
*-2 dilution plate: spreading of *Azospirillum brasilense* sp7 having the pCZ-exaA-*acdS* construct onto LBA plate ( with Ampicillin, the selective marker for Azospirillum and tetracycline, the selective marker for pCZ plasmid present in the media)*

![](https://static.igem.wiki/teams/4392/wiki/results/3dilution-igem.jpg)
*-3 dilution plate: spreading of *Azospirillum brasilense* sp7 having the pCZ-exaA-*acdS* construct onto LBA plate ( with Ampicillin, the selective marker for Azospirillum and tetracycline, the selective marker for pCZ plasmid present in the media). The single colonies were obtained from this plate*

![](https://static.igem.wiki/teams/4392/wiki/results/conjugatedplate-igem.jpg)
*Streaking of *Azospirillum brasilense* sp7 with the pCZ-exaA-*acdS* construct onto LBA plate ( with Ampicillin, the selective marker for Azospirillum and tetracycline, the selective marker for pCZ plasmid present in the media)*

### 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

 ![](https://static.igem.wiki/teams/4392/wiki/results/clones/failed-fdhf.jpg)
 *All the *fdhF* promoter bands in the gradient PCR were masked*

![](https://static.igem.wiki/teams/4392/wiki/results/clones/fdhf-amplified.jpg)
 *The gradient PCR showed that 48°C is the ideal annealing temperature*

![](https://static.igem.wiki/teams/4392/wiki/results/clones/amplified-fdhf.jpg)
 *Amplified *fdhF* promoter after using the modified dye*

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

 ![](https://static.igem.wiki/teams/4392/wiki/results/clones/colony-pcr-fdhf.jpg)
 *Colony PCR results in 1.5% agarose gel*

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.

![](https://static.igem.wiki/teams/4392/wiki/results/clones/native-page.jpg)
 *Native PAGE Results*

### 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.

 ![](https://static.igem.wiki/teams/4392/wiki/results/clones/gradient-pcr-lipo-pak.jpg)
 *Gradient PCR of acdS gene*

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.

 ![](https://static.igem.wiki/teams/4392/wiki/results/clones/pcr-plasmid-extraction-pak-acds.jpg)
 *Extracted plasmids in 1% agarose gel*

### References

[1] Part:BBa\_K387003[http://parts.igem.org/Part:BBa\_K387003](https://doi.org/10.1128/JB.00035-17)

[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](https://2022.igem.wiki/iiser-pune-india/proof-of-concept#hi2) 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](https://2022.igem.wiki/iiser-pune-india/proof-of-concept#hi2) 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.

 ![](https://static.igem.wiki/teams/4392/wiki/results/phenol-red-lba-plate-with-azospirillum.jpg)
 *An example of a Phenol Red+ LBA plate with Azospirillum*

#### 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

 ![](https://static.igem.wiki/teams/4392/wiki/results/growth-curves/growth-curves-1.jpg)

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

 ![](https://static.igem.wiki/teams/4392/wiki/results/growth-curves/a-br-wt-nfb.png)

*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

![](https://static.igem.wiki/teams/4392/wiki/results/growth-curves/a-br-wt-nfb-0-25-g-l.png)

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.

 ![](https://static.igem.wiki/teams/4392/wiki/results/growth-curves/a-br-wt-growth-curve-nfb-0-5g-l.png)

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.

[← Notebook](https://2022.igem.wiki/iiser-pune-india/notebook)
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