iGEM 2019 — Team IIT-Madras

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# Team:IIT-Madras/Notebook

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People

[Team](https://2019.igem.org/Team%3AIIT-Madras/Team)
[Collaborations](https://2019.igem.org/Team%3AIIT-Madras/Collaborations)
[Attributions](https://2019.igem.org/Team%3AIIT-Madras/Attributions)

Project

[Simple](https://2019.igem.org/Team%3AIIT-Madras/Simple)
[Meet our Chassis](https://2019.igem.org/Team%3AIIT-Madras/Chassis)
[Description](https://2019.igem.org/Team%3AIIT-Madras/Description)
[Design](https://2019.igem.org/Team%3AIIT-Madras/Design)
[Protocols](https://static.igem.org/mediawiki/2019/4/4e/T--IIT-Madras--protocols.pdf)
[Safety](https://2019.igem.org/Team%3AIIT-Madras/Safety)
[Notebook](https://2019.igem.org/Team%3AIIT-Madras/Notebook)
[Results](https://2019.igem.org/Team%3AIIT-Madras/Results)

[Parts](https://2019.igem.org/Team%3AIIT-Madras/Parts)
[Software](https://2019.igem.org/Team%3AIIT-Madras/Software)
[Judging](https://igem.org/2019_Judging_Form?team=IIT-Madras)

The Language Project

[Journey So Far](https://2019.igem.org/Team%3AIIT-Madras/The_Language_Project)
[Language Project 2.0](https://2019.igem.org/Team%3AIIT-Madras/LP2.0)
[Comic Strips](https://2019.igem.org/Team%3AIIT-Madras/Comic_Strips)

Human Practices

[Integrated Human Practices](https://2019.igem.org/Team%3AIIT-Madras/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3AIIT-Madras/Public_Engagement)

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[Simple](https://2019.igem.org/Team%3AIIT-Madras/Simple)
[Project](https://2019.igem.org/Team%3AIIT-Madras/Description)
[Team](https://2019.igem.org/Team%3AIIT-Madras/Team)
[The Language Project](https://2019.igem.org/Team%3AIIT-Madras/The_Language_Project)
[Human Practices](https://2019.igem.org/Team%3AIIT-Madras/Human_Practices)
[Site Map](https://2019.igem.org/Team%3AIIT-Madras/Site_Map)

# NOTEBOOK

2019

MAY
Week 3

Glycerol stock of *Fusarium solani* was collected from Professor Smita Srivastava’s lab.

We attempted making Potato Dextrose Agar (PDA) from scratch using potato infusion dextrose and agar. We poured slants with this media and inoculated *Fusarium solani* on them.

MAY
Week 4

More slants were inoculated.

We sought help from Professor S.N. Gummadi and his lab members about fungal transformation.

We made glycerol stocks of the fungal spores and stored them at -80 degrees.

JUNE
Week 1

Several glycerol stocks of *F. solani* were made and stored.

JUNE
Week 2

We discussed the protocol for fungal transformation with Professor S.N. Gummadi and Professor Smita Srivastava and decided upon using an electroporation mediated transformation

We prepared the solutions required for transformation.

JUNE
Week 3

We inoculated TOP10 containing pCAMBIA 1301 for plasmid isolation.

We discussed the usage of the enzyme beta-glucuronidase in fungal transformation and sought help with plasmid isolation from Professor Nitish R Mahapatra.

With the help of Vikas and Dhanya from Professor Nitish’s lab, we isolated pCAMBIA 1301.

JUNE
Week 4

Khwajah from Professor Smita Srivastava’s lab suggested that we incubate slants for 7 days, collect the spores, grow them in liquid medium and then perform transformation.

We did a second round of plasmid isolation.

We poured slants, inoculated them with spores and incubated them for 7 days.

JULY
Week 1

We discussed plasmid design with Vikas from Prof. Nitish’s lab. We inoculated the spores in liquid broth. We plated *E. coli* TOP10 for iGEM REC team.

Constructs of STR and GFP were designed and ordered from Twist Bioscience and IDT.

JULY
Week 2

We performed electroporation based transformation and plated the transformants using kanamycin as the selectable marker. The control plate had no DNA.

There was growth on all the plates. However, kanamycin cannot be used a selectable marker for transformation in fungi as it’s expressed under a promoter specific to *E. coli*.

We corrected this by using hygromycin as a selectable marker in all further transformations.

JULY
Week 3

Another liquid culture was inoculated for transformation. Transformants were selected for on hygromycin plates. These colonies were isolated and streaked on plates for GUS staining to confirm transformation.

JULY
Week 4

We checked whether the usage of beta-glucuronidase would improve the efficiency of transformation. We sought inputs from Prof. Subramaniam on this.

The next round of transformation was conducted in two batches - with and without beta-glucuronidase. This time the transformants were directly plated for GUS staining.

AUG
Week 1

Fungal plasmid isolation was performed.

Gel electrophoresis was performed with the isolated plasmids. The gel didn’t show anything apart from the ladder.

We repeated transformation with beta-glucuronidase as the previous transformation failed. We plated on hygromycin.

We inoculated fungi for another round of transformation.

AUG
Week 2

We realised that we failed to add SDS in our previous plasmid isolation from fungi which resulted in no lysis.

Another culture inoculated for transformation.

AUG
Week 3

Transformation was carried out again.

We prepared reagents for another transformation.

We isolated plasmid from a transformed fungal culture using a different protocol under Khwajah's guidance.

AUG
Week 4

We ran a gel with our isolated plasmid samples. It was empty.

We performed another round of plasmid isolation and did not succeed.

The ordered parts arrived! Hurrah!

SEPT
Week 1

Our constructs were designed to compatible with Gibson assembly and restriction based cloning. There is no lab in the institute to guide us with Gibson assembly, so we decided to clone the parts into pCAMBIA 1301 using restriction enzymes.

Prof. Nitish's lab guided us with cloning. Abrar, Anand, Dhanya, and Vikas guided us.

SEPT
Week 2

We isolated plasmids for digestion using a mini-prep.

We set up restriction digestion for GFP (using PstI and AflII), STR (using EcoRI and PstI) and pCAMBIA 1301 (using AflII and EcoRI).

The GFP and STR fragments were ligated using a T4 ligase. The fragment was then ligated with the cut plasmid.

The ligation mixture was transformed into competent TOP10 cells.

We got one colony.  *One colony!*

We ordered primers for GFP and STR to amplify the parts.

SEPT
Week 3

The colony was inoculated in 5ml of LB and plasmid isolation was performed from 2ml of this culture. The isolates were run on a gel but sadly, no bands were observed.

Cloning had failed and we had to rethink our strategy.

SEPT
Week 4

We redid plasmid isolation from *E.coli* transformed with our part from the remaining liquid culture. A smear was observed on the gel.

Our anxiety peaked while waiting for primers to arrive.

OCT
Week 1

Primers arrived!

We reconstituted primers and set up a gradient PCR for GFP and STR to find the annealing temperatures.

PCRs for GFP and STR were set up using the annealing temperatures obtained. These were purified using a PCR cleanup.

We used the same cloning strategy and repeated restriction digestion, ligation and transformation.

This failed.

We had a fruitful discussion with Prof. Subramaniam and Prof. Nitish who helped us redesign our cloning strategy.

OCT
Week 2

We started all the procedures for cloning from the start. Fun times.

Our plasmid quality was low, so we isolated plasmid using a midi-prep. GFP was amplified and gel purified.

We digested GFP and pCAMBIA 1301 followed by ligation and transformation into ultra-competent DH5α cells.

OCT
Week 2

Success!

We had over 25 colonies. These were inoculated in LB and plasmids were isolated. This was confirmed by running a gel.

These plasmids were subjected to restriction digestion to observe an insert release.

This part of GFP in pCAMBIA 1301 under the control of a CaMV35s promoter was validated by fluorometry studies.

OCT
Week 3

We characterised a CaMV35s promoter in *E. coli* using a hph gene attached to the promoter. The MIC of *E. coli* TOP10 was found to be 50µg/ml. TOP10 transformed with the above part was resistant to up to 200µg/ml of Hygromycin.

Contact Us

Department of Biotechnology, IIT Madras.
Chennai 36, India.

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Sponsors

[![](https://static.igem.org/mediawiki/2019/e/e1/T--IIT-Madras--MFF_logo.jpeg)](https://www.mehtafamilyfoundation.org/)

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# Team:IIT-Madras/Experiments

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[HOME](https://2019.igem.org/Team%3AIIT-Madras)

TEAM

[Team Members](https://2019.igem.org/Team%3AIIT-Madras/Team)
[Collaborations](https://2019.igem.org/Team%3AIIT-Madras/Collaborations)

PROJECT

[Description](https://2019.igem.org/Team%3AIIT-Madras/Description)
[Design](https://2019.igem.org/Team%3AIIT-Madras/Design)
[Experiments](https://2019.igem.org/Team%3AIIT-Madras/Experiments)
[Notebook](https://2019.igem.org/Team%3AIIT-Madras/Notebook)
[Contribution](https://2019.igem.org/Team%3AIIT-Madras/Contribution)
[Results](https://2019.igem.org/Team%3AIIT-Madras/Results)
[Demonstrate](https://2019.igem.org/Team%3AIIT-Madras/Demonstrate)
[Improve](https://2019.igem.org/Team%3AIIT-Madras/Improve)
[Attributions](https://2019.igem.org/Team%3AIIT-Madras/Attributions)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AIIT-Madras/Parts)
[Basic Parts](https://2019.igem.org/Team%3AIIT-Madras/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AIIT-Madras/Composite_Part)
[Part Collection](https://2019.igem.org/Team%3AIIT-Madras/Part_Collection)

[SAFETY](https://2019.igem.org/Team%3AIIT-Madras/Safety)

HUMAN PRACTICES

[Human Practices](https://2019.igem.org/Team%3AIIT-Madras/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AIIT-Madras/Public_Engagement)

AWARDS

[Entrepreneurship](https://2019.igem.org/Team%3AIIT-Madras/Entrepreneurship)
[Hardware](https://2019.igem.org/Team%3AIIT-Madras/Hardware)
[Measurement](https://2019.igem.org/Team%3AIIT-Madras/Measurement)
[Model](https://2019.igem.org/Team%3AIIT-Madras/Model)
[Plant](https://2019.igem.org/Team%3AIIT-Madras/Plant)
[Software](https://2019.igem.org/Team%3AIIT-Madras/Software)

[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=IIT-Madras)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:IIT-Madras/Results

[![oops there ought to be a logo here](https://static.igem.org/mediawiki/2019/8/8c/T--IIT-Madras--logo_rectagle_transparent.png)](https://2019.igem.org/Team%3AIIT-Madras)

People

[Team](https://2019.igem.org/Team%3AIIT-Madras/Team)
[Collaborations](https://2019.igem.org/Team%3AIIT-Madras/Collaborations)
[Attributions](https://2019.igem.org/Team%3AIIT-Madras/Attributions)

Project

[Simple](https://2019.igem.org/Team%3AIIT-Madras/Simple)
[Meet our Chassis](https://2019.igem.org/Team%3AIIT-Madras/Chassis)
[Description](https://2019.igem.org/Team%3AIIT-Madras/Description)
[Design](https://2019.igem.org/Team%3AIIT-Madras/Design)
[Protocols](https://static.igem.org/mediawiki/2019/4/4e/T--IIT-Madras--protocols.pdf)
[Safety](https://2019.igem.org/Team%3AIIT-Madras/Safety)
[Notebook](https://2019.igem.org/Team%3AIIT-Madras/Notebook)
[Results](https://2019.igem.org/Team%3AIIT-Madras/Results)

[Parts](https://2019.igem.org/Team%3AIIT-Madras/Parts)
[Software](https://2019.igem.org/Team%3AIIT-Madras/Software)
[Judging](https://igem.org/2019_Judging_Form?team=IIT-Madras)

The Language Project

[Journey So Far](https://2019.igem.org/Team%3AIIT-Madras/The_Language_Project)
[Language Project 2.0](https://2019.igem.org/Team%3AIIT-Madras/LP2.0)
[Comic Strips](https://2019.igem.org/Team%3AIIT-Madras/Comic_Strips)

Human Practices

[Integrated Human Practices](https://2019.igem.org/Team%3AIIT-Madras/Human_Practices)
[Public Engagement](https://2019.igem.org/Team%3AIIT-Madras/Public_Engagement)

[![oops there ought to be a logo here](https://static.igem.org/mediawiki/2019/8/8c/T--IIT-Madras--logo_rectagle_transparent.png)](https://2019.igem.org/Team%3AIIT-Madras)

[Simple](https://2019.igem.org/Team%3AIIT-Madras/Simple)
[Project](https://2019.igem.org/Team%3AIIT-Madras/Description)
[Team](https://2019.igem.org/Team%3AIIT-Madras/Team)
[The Language Project](https://2019.igem.org/Team%3AIIT-Madras/The_Language_Project)
[Human Practices](https://2019.igem.org/Team%3AIIT-Madras/Human_Practices)
[Site Map](https://2019.igem.org/Team%3AIIT-Madras/Site_Map)

# RESULTS

## *F. solani* Growth

The fungus in liquid culture is incubated at 27°C and 120 rpm for 7 days.

## Optimisation of *F. solani* Transformation

The plasmid pCAMBIA 1301 contains the GUS reporter system and the antibiotic resistance genes for hygromycin and kanamycin. For transformation of this into *F. solani*, the protocol adopted was electroporation.

Fungal culture along with DNA and a negative control with no DNA were subjected to electroporation. They were subsequently streaked onto hygromycin B slants. After a few days there was no growth in the negative control as expected and the other slant showed growth indicating successful transformation.

![](https://static.igem.org/mediawiki/2019/f/f6/T--IIT-Madras--Fsolanislants.jpeg)

*Figure 1: Slants with the transformed cultures*

## Cloning of BioBricks

The first step in the cloning of our BioBricks into the plasmid pCAMBIA 1301 was the isolation of the plasmid from a stock *E. coli* TOP10 culture. This glycerol stock was plated and then inoculated in LB medium from which plasmids were extracted through a kit-free midiprep. The isolation of plasmids was confirmed through agarose gel electrophoresis. The InvitrogenTM 1 Kb Plus DNA Ladder was used in all electrophoresis experiments.

![](https://static.igem.org/mediawiki/2019/7/7c/T--IIT-Madras--midiprep.png)

*Figure 2: pCAMBIA 1301 Isolated through Midiprep*

The isolated plasmids were then subjected to a double-digestion using the restriction enzymes PstI and AflII so the eGFP construct could be cloned into it. Restriction digestion was seen to have worked and the band of the right size (8,968 bp) was excised from the gel and eluted.

![](https://static.igem.org/mediawiki/2019/4/4a/T--IIT-Madras--pCAMBIAdigestion.png)

*Figure 3: Double-Digestion of pCAMBIA 1301*

The two inserts containing eGFP and the enzyme strictosidine synthase (STR) respectively were PCR-amplified from the product obtained from IDT. The PCR products were run on a gel to confirm amplification. Having confirmed that amplification had worked, both products were subjected to double-digestion. The construct containing STR was digested with EcoRI and PstI while the construct containing eGFP was digested with PstI and AflII. All restriction enzymes were obtained from New England Biolabs.

![](https://static.igem.org/mediawiki/2019/0/0a/T--IIT-Madras--InsertAmpli.png)

*Figure 4: PCR-Amplification of the Inserts*

An overnight ligation reaction using T4 DNA Ligase was set up in which the eGFP insert was cloned into the plasmid backbone. The reaction mixture was then transformed into ultracompetent *E. coli* TOP10 cells which were then plated on LB Media containing kanamycin (50 ug/ml) and incubated overnight. This yielded over 25 colonies. A plasmid self-ligation was set up as a negative control. This reaction mixture, when transformed into ultracometent TOP10 cells, yielded no colonies that survived on media containing kanamycin.

Liquid cultures of 10 tranformant colonies were set up and plasmids were isolated from them through a miniprep protocol. The presence of plasmids was confirmed through agarose gel electrophoresis.

![](https://static.igem.org/mediawiki/2019/b/b3/T--IIT-Madras--ClonedPlasmid.png)

*Figure 5: Plasmids Isolated from 10 Transformant Colonies*

To confirm that the isolated plasmids contained the eGFP insert, double-digestions were set up in which the plasmids isolated from each colony were digested using PstI and AflII so as to release the insert. The resulting reaction mixture was run on a gel to confirm the presense of the insert.

![](https://static.igem.org/mediawiki/2019/2/20/T--IIT-Madras--ClonedDigestion.png)

*Figure 6: Insert Release to Confirm Cloning*

For further confirmation, PCR reactions were set up using plasmids isolated from each colony as the template and forward and reverse primers specific to the insert. Bright bands were observed for all colonies.

![](https://static.igem.org/mediawiki/2019/b/b4/T--IIT-Madras--ClonedPCR.png)

*Figure 6: PCR to Confirm Cloning*

## Part Characterisation

INTRODUCTION:

The Cauliflower Mosaic Virus (CaMV) promoter 35S is a strong constitutive plant promoter (Odell et al. 1985; Benfey and Chua 1989). Studies indicate that this promoter can be used to express genes in *E. coli*.( Assaad and Signer 1990). This can be advantageous as expression can be verified rapidly and with considerable saving of time and manipulation as this promoter is typically used with plant constructs.

We have validated the expression of an enhanced CaMV 35S promoter fused to a hph gene cassette in *E. coli*. This is a part of the plant vector pCAMBIA 1301 which has been used for our experiments. Details of the usage of this plasmid can be found  [here.](https://2019.igem.org/Team%3AIIT-Madras/Design)

MATERIALS AND METHODS:

TOP 10 with the pCAMBIA 1301 plasmid was grown on LB agar. 100 ul of an overnight culture (5 mL) developed from a single colony was used to plate on 10 plates with varying concentrations of hygromycin B (100-1000 ug/ml).

RESULTS:

*E. coli* had no visible growth on 50 ug/ml of hygromycin B and this was found to be the minimum inhibitory concentration. This value is consistent with other studies.
Our transformed cells containing the hph gene conferring resistance to hygromycin B under the control of the CaMV 35s promoter were resistant to up to 200 ug/ml of hygromycin B.
![](https://static.igem.org/mediawiki/2019/1/19/T--IIT-Madras--100hyg.jpeg)

*Figure 7: TOP 10 + pCAMBIA 1301 in 100ug/ml of Hygromycin*

![](https://static.igem.org/mediawiki/2019/4/42/T--IIT-Madras--150hyg.jpeg)

*Figure 8: TOP 10 + pCAMBIA 1301 in 150ug/ml of Hygromycin*

![](https://static.igem.org/mediawiki/2019/a/af/T--IIT-Madras--200hyg.jpeg)

*Figure 9: TOP 10 + pCAMBIA 1301 in 200ug/ml of Hygromycin*

![](https://static.igem.org/mediawiki/2019/0/05/T--IIT-Madras--allhygplates.jpeg)

*Figure 10: TOP 10 + pCAMBIA 1301 in 300-1000ug/ml of Hygromycin*

CALCULATIONS:

We calculated the initial number of cells to be 7.2 x 108 cells/ml based on the OD.

At 50ug/ml of hygromycin B, the number of cells =7.2 x 108 cells/ml

At 100ug/ml of hygromycin B, the number of cells=7.2 x 108 cells/ml

At 200ug/ml of hygromycin B, the number of cells=(say 1/5th of what we had earlier) 9 x 107 cells/ml

![](https://static.igem.org/mediawiki/2019/0/0c/T--IIT-Madras--hygsensitivityassay.png)

*Figure 11: %Sensitivity of cells in response to varying hygromycin B concentration*

## Part Validation

We created a part ( BBa\_K3163002) such that eGFP is driven by a CaMV 35s Promoter and flanked by NOS terminator which is already present in pCAMBIA 1301. We cloned this part into pCAMBIA which replicates in Fusarium solani and E. coli. Here, we have transformed the plasmid in to E. coli TOP 10 and performed fluorometry studies to confirm the presence of GFP. analysis. Higher fluorescence in the culture containing our part confirmed the presence of GFP and this was found to be statistically significant by ANOVA analysis. Characterisation of BBa\_K3163002 in E. coli is reported in the figure given below.

![](https://static.igem.org/mediawiki/2019/6/64/T--IIT-Madras--Silver_characterization.jpeg)
*Figure 12: Normalized Fluorescence with respect to Empty Vector (pCAMBIA 1301) in E. coli.*

Contact Us

Department of Biotechnology, IIT Madras.
Chennai 36, India.

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Sponsors

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