iGEM 2019 — Team IISc-Bangalore

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# Team:IISc-Bangalore/Notebook

[![](https://static.igem.org/mediawiki/2019/0/0c/T--IISc-Bangalore--horizontal-logo.png)](https://2019.igem.org/Team%3AIISc-Bangalore)

* Project

  [Description & Inspiration](https://2019.igem.org/Team%3AIISc-Bangalore/Description)
  [Design](https://2019.igem.org/Team%3AIISc-Bangalore/Design)
  [Experiments](https://2019.igem.org/Team%3AIISc-Bangalore/Experiments)
  [Parts](https://2019.igem.org/Team%3AIISc-Bangalore/Parts)
  [Modelling](https://2019.igem.org/Team%3AIISc-Bangalore/Model)
* Lab

  [Protocols](https://2019.igem.org/Team%3AIISc-Bangalore/Lab/Protocol)
  [Growth Regulation](https://2019.igem.org/Team%3AIISc-Bangalore/Growth_Regulation)
  [Assembly](https://2019.igem.org/Team%3AIISc-Bangalore/Assembly)
  [Characterisation](https://2019.igem.org/Team%3AIISc-Bangalore/Characterisation)
  [Improvement](https://2019.igem.org/Team%3AIISc-Bangalore/Improve)
  [Results](https://2019.igem.org/Team%3AIISc-Bangalore/Results)
  [Lab Notebook](https://2019.igem.org/Team%3AIISc-Bangalore/Notebook)
  [Safety](https://2019.igem.org/Team%3AIISc-Bangalore/Safety)
* [Hardware](https://2019.igem.org/Team%3AIISc-Bangalore/Hardware)

  [Introduction](https://2019.igem.org/Team%3AIISc-Bangalore/Hardware)
  [OptoMatic](https://2019.igem.org/Team%3AIISc-Bangalore/OptoMatic)
  [Impedance Method](https://2019.igem.org/Team%3AIISc-Bangalore/OptoMatic/Impedance)
  [Absorbance Method](https://2019.igem.org/Team%3AIISc-Bangalore/OptoMatic/Absorbance)
  [Zeta Potential Method](https://2019.igem.org/Team%3AIISc-Bangalore/OptoMatic/Zeta)
* [Software](https://2019.igem.org/Team%3AIISc-Bangalore/Software)
* Community

  [Collaborations](https://2019.igem.org/Team%3AIISc-Bangalore/Collaborations)
  [Human Practices](https://2019.igem.org/Team%3AIISc-Bangalore/Human_Practices)
  [Integrated Human Practices](https://2019.igem.org/Team%3AIISc-Bangalore/Integrated_HP)
  [Public Engagement](https://2019.igem.org/Team%3AIISc-Bangalore/Public_Engagement)
* Team

  [Philosophy](https://2019.igem.org/Team%3AIISc-Bangalore/Philosophy)
  [Members](https://2019.igem.org/Team%3AIISc-Bangalore/Team)
  [Attributions](https://2019.igem.org/Team%3AIISc-Bangalore/Attributions)

# Laboratory Notebook

###### Quick links

* Download the Complete Notebook

This page contains a weekly account of the experiments and activities conducted in the laboratory.

Week 1
Specific buffers were made and autoclaved. Prepared antibiotic solutions and poured plates. DH5-α competent cells were prepared, and PCR mixes were made for 4 different DNA sequences and 5 different controls. Transformant bacteria (RFP) were created. *E. coli* was streaked.

Week 2
LB was autoclaved, and plates were spread. Absorbance values for *E. coli* and *B. cereus* was measured. Antibiotic solutions of Ampicillin and Chloramphenicol were made.

Week 3
Gel Electrophoresis for CcaS-PCR, was performed. PCR was performed for the miniprepped products of ECFP-1 and ECFP-2. Bba\_K3165039 was ordered. Bba\_K3165041 and Bba\_K3165036 was ordered for CcaS system. For Opto-T7 system, Bba\_K3165050, Bba\_K3165052 and Bba\_K3165054 was ordered.

Week 4
Gradient PC/h6>Rs for PMag-gP2 was carried out with different initial reagent concentrations. PCR for CcaS system was also performed and PCR, for CcaR and Cyan was performed. PCRs for Opto-T7 NMag and touchdown PCR for CCaR and Cyan were performed. Miniprep for pBS1C was performed.

Week 5
Restriction digestion of pBS1C and pBS4S was performed. Cloning trial for Opto-T7 was performed. Restriction digestion was performed on the following plasmids: pBS4S, pBS1C and PMag. SDS-Page for ECF-P was performed. Double digest with EcoRI and PstI was performed. Midiprep for pSB1C3 was performed. pBS4S and pBS1c were ligated.

Week 6
Digestion on last week's midiprep was performed. First attempt for ligation of pSB1C3 with CcaR was performed. pBS1C and pBS4S were restriction digested for E and P.

Week 7
Ligation was attempted for the second time. Miniprep was performed on 8 of the chosen colonies which was obtained for the aforementioned ligation. Gel extraction for pBS1C and pBS4S was performed. Double digestion for CcaR ligation for 18 colonies was carried out.

Week 8
Gel electrophoresis was carried out, from 10 of the CcaR-digested colonies and PCR was carried out on the DNA from the colonies. ECFP characterization was attempted.

Week 9
PCR for mCherry and i2mCherry(Leu) was carried out. The DNA concentration for the above parts were found out through NanoDropTM. A gradient PCR for improvement of parts was done.

Week 10
The digested pSB1C3 from RFP-Biobrick and the RFP fragment were purified by the standard agarose gel-extraction procedure. Ligation for improvement-part was carried out.

Week 11
The ligated product was transformed. Digesition for pSB1C3(ECFP), miniprepped, i2mCherry(Ile) PCRed product and i2mCherry(Leu)-pSB1C3 ligated product was obtained.

Week 12
Improvement of pSB1C3 for restriction sites E and S was carried out. Gel extraction was carried out in an attempt to extract the required plasmid.

To view a detailed account of our laboratory activities, download the PDF attached below.

[![](https://static.igem.org/mediawiki/2018/1/1c/T--IISc-Bangalore--download.png) Download Complete Lab Notebook](https://static.igem.org/mediawiki/2019/3/34/T--IISc-Bangalore--Wetlab_Notebook.pdf)

[![](https://static.igem.org/mediawiki/2019/thumb/1/1d/T--IISc-Bangalore--Fb_g.png/120px-T--IISc-Bangalore--Fb_g.png)](https://www.facebook.com/iiscbangaloreigem/)

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[![](https://static.igem.org/mediawiki/2019/archive/f/f9/20191021164133%21T--IISc-Bangalore--IISc_fc2.png)
![](https://static.igem.org/mediawiki/2019/archive/f/f9/20191021171842%21T--IISc-Bangalore--IISc_fc2.png)](https://www.iisc.ac.in/)

[![](https://static.igem.org/mediawiki/2019/archive/e/e6/20191021163311%21T--IISc-Bangalore--IDT_fc2.png)
![](https://static.igem.org/mediawiki/2019/archive/e/e6/20191021163319%21T--IISc-Bangalore--IDT_fc2.png)](https://eu.idtdna.com/pages)

[![](https://static.igem.org/mediawiki/2019/b/bf/T--IISc-Bangalore--DBT_g2.png)
![](https://static.igem.org/mediawiki/2019/d/d5/T--IISc-Bangalore--DBT_fc2.png)](http://dbtindia.gov.in/)

[![](https://static.igem.org/mediawiki/2019/archive/a/a1/20191021163502%21T--IISc-Bangalore--NewEngBL_fc2.png)
![](https://static.igem.org/mediawiki/2019/archive/a/a1/20191021163455%21T--IISc-Bangalore--NewEngBL_fc2.png)](https://international.neb.com/)

[![](https://static.igem.org/mediawiki/2019/archive/6/64/20191021162740%21T--IISc-Bangalore--SnapGene_g2.png)
![](https://static.igem.org/mediawiki/2019/archive/6/68/20191021163106%21T--IISc-Bangalore--SnapGene_fc2.png)](https://www.snapgene.com/)

[![](https://static.igem.org/mediawiki/2019/archive/d/d6/20191021163617%21T--IISc-Bangalore--Twist_fc2.png)
![](https://static.igem.org/mediawiki/2019/archive/d/d6/20191021163611%21T--IISc-Bangalore--Twist_fc2.png)](https://twistbioscience.com/)

[![](https://static.igem.org/mediawiki/2019/archive/9/94/20191021163730%21T--IISc-Bangalore--MathWorks_fc2.png)
![](https://static.igem.org/mediawiki/2019/archive/9/94/20191021163722%21T--IISc-Bangalore--MathWorks_fc2.png)](https://in.mathworks.com/)

[![](https://static.igem.org/mediawiki/2019/archive/b/b3/20191021162839%21T--IISc-Bangalore--GenScript_g2.png)
![](https://static.igem.org/mediawiki/2019/archive/f/f4/20191021163004%21T--IISc-Bangalore--GenScript_fc2.png)](https://www.genscript.com/)

[![](https://static.igem.org/mediawiki/2019/archive/a/a4/20191021162515%21T--IISc-Bangalore--Benchling_g2.png)
![](https://static.igem.org/mediawiki/2019/archive/0/04/20191021162240%21T--IISc-Bangalore--Benchling_fc2.png)](https://www.benchling.com/)

Links

[Address](https://goo.gl/maps/BPKKwKU3hd32)

Contact

Best Viewed on Desktop


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:IISc-Bangalore/Experiments

[![](https://static.igem.org/mediawiki/2019/0/0c/T--IISc-Bangalore--horizontal-logo.png)](https://2019.igem.org/Team%3AIISc-Bangalore)

* Project

  [Description & Inspiration](https://2019.igem.org/Team%3AIISc-Bangalore/Description)
  [Design](https://2019.igem.org/Team%3AIISc-Bangalore/Design)
  [Experiments](https://2019.igem.org/Team%3AIISc-Bangalore/Experiments)
  [Parts](https://2019.igem.org/Team%3AIISc-Bangalore/Parts)
  [Modelling](https://2019.igem.org/Team%3AIISc-Bangalore/Model)
* Lab

  [Protocols](https://2019.igem.org/Team%3AIISc-Bangalore/Lab/Protocol)
  [Growth Regulation](https://2019.igem.org/Team%3AIISc-Bangalore/Growth_Regulation)
  [Assembly](https://2019.igem.org/Team%3AIISc-Bangalore/Assembly)
  [Characterisation](https://2019.igem.org/Team%3AIISc-Bangalore/Characterisation)
  [Improvement](https://2019.igem.org/Team%3AIISc-Bangalore/Improve)
  [Results](https://2019.igem.org/Team%3AIISc-Bangalore/Results)
  [Lab Notebook](https://2019.igem.org/Team%3AIISc-Bangalore/Notebook)
  [Safety](https://2019.igem.org/Team%3AIISc-Bangalore/Safety)
* [Hardware](https://2019.igem.org/Team%3AIISc-Bangalore/Hardware)

  [Introduction](https://2019.igem.org/Team%3AIISc-Bangalore/Hardware)
  [OptoMatic](https://2019.igem.org/Team%3AIISc-Bangalore/OptoMatic)
  [Impedance Method](https://2019.igem.org/Team%3AIISc-Bangalore/OptoMatic/Impedance)
  [Absorbance Method](https://2019.igem.org/Team%3AIISc-Bangalore/OptoMatic/Absorbance)
  [Zeta Potential Method](https://2019.igem.org/Team%3AIISc-Bangalore/OptoMatic/Zeta)
* [Software](https://2019.igem.org/Team%3AIISc-Bangalore/Software)
* Community

  [Collaborations](https://2019.igem.org/Team%3AIISc-Bangalore/Collaborations)
  [Human Practices](https://2019.igem.org/Team%3AIISc-Bangalore/Human_Practices)
  [Integrated Human Practices](https://2019.igem.org/Team%3AIISc-Bangalore/Integrated_HP)
  [Public Engagement](https://2019.igem.org/Team%3AIISc-Bangalore/Public_Engagement)
* Team

  [Philosophy](https://2019.igem.org/Team%3AIISc-Bangalore/Philosophy)
  [Members](https://2019.igem.org/Team%3AIISc-Bangalore/Team)
  [Attributions](https://2019.igem.org/Team%3AIISc-Bangalore/Attributions)

# Experiments

###### Quick links

* Assembling parts of the CcaS/R and OPTO-T7 systems

* ECFP Characterisation

* Protein Precipitation Assay
* Fluorescence measurement

* Truncation Assay

* Experiments on ccdB gene system

* Growth assay
* Protein extraction and purification
* Protein Thermal Shift assay

* Potential future experiment

## Assembling parts of the CcaS/R and Opto-T7 systems:

Molecular cloning methods were performed to bring together the different gene fragments of the [CcaS/R system](http://parts.igem.org/Part%3ABBa_K3165042) and the [Opto-T7 system](http://parts.igem.org/Part%3ABBa_K3165055).

[![Molecular cloning](https://static.igem.org/mediawiki/2019/7/78/T--IISc-Bangalore--Molecular_cloning_final.png)](https://static.igem.org/mediawiki/2019/7/78/T--IISc-Bangalore--Molecular_cloning_final.png)

Fig.1-Molecular cloning

## ECFP Characterization:

Since we used [ECFP](http://parts.igem.org/Part%3ABBa_K3165006) as a reporter protein in our CcaS/R system, we characterized it using the following experiments:

1. **Protein Precipitation Assay:**

   For the purification of a protein, one nice method is the Ammonium Sulphate Precipitation method in which different proteins precipitate out from the protein lysate solution at different salt concentrations, i.e. different proteins “salt out” at different salt ((NH4)2SO4) concentrations. To determine the salt concentration at which ECFP protein precipitates, we performed the Ammonium Sulphate Precipitation Assay ([Protocol page](https://2019.igem.org/Team%3AIISc-Bangalore/Lab/Protocol)).

   The results for this can be found on the [Result page](https://2019.igem.org/Team%3AIISc-Bangalore/Results).

   [![Protein precipitation assay](https://static.igem.org/mediawiki/2019/b/b2/T--IISc-Bangalore--AMMONIUM_SULPHATE_FINAL1.png)](https://static.igem.org/mediawiki/2019/b/b2/T--IISc-Bangalore--AMMONIUM_SULPHATE_FINAL1.png)

   Fig.2-Ammonium sulphate protein precipitation assay
2. **Fluorescence measurement:**

   To determine the absorption and emission maxima of our fluorophore ECFP, we did a wavelength scan and observed the fluorescence intensity peaks.The fluorescence measurements can be found on the [Result page](https://2019.igem.org/Team%3AIISc-Bangalore/Results).

## Truncation Assay:

To quantify the truncation due to the internal RBS in [i2mCherry (Leu)](http://parts.igem.org/Part%3ABBa_K3165049), [i2mCherry (Ile)](http://parts.igem.org/Part%3ABBa_K3165046) and [mCherry](http://parts.igem.org/Part%3ABBa_J18932), we used the following two strategies:

1. Firstly, a rough estimate was obtained by comparing the band intensities directly from the gel electrophoresis of the total cell lysate after expressing the protein.
2. For a more accurate quantification, we purified the protein using Ni-NTA and measured the fluorescence obtained in all (supernatant, wash, and elution) fractions. In an ideal case, the ratio of fluorescence obtained in the supernatant and elution fractions would give the fraction of protein that was truncated (assuming truncated and non-truncated i2mCherry (Leu), i2mCherry (Ile) and mCherry are equally fluorescent). However, the process of binding of 6xHis to Ni-NTA is not perfect and proteins tend to run off into the supernatant and the washes. To get rid of this experimental error, we performed PAGE with each fraction and divided the total fluorescence of the fraction into two parts - truncated/non-truncated - using the band intensities. The fluorescence for the truncated protein from each fraction was then summed up and divided by the fluorescence of the non-truncated part to get the relative fractions of the two varieties. Concerned results can be found on the [Result page](https://2019.igem.org/Team%3AIISc-Bangalore/Results).

   [![Ni-NTA protein purification assay](https://static.igem.org/mediawiki/2019/b/b4/T--IISc-Bangalore--Ni-NTA_purification.png)](https://static.igem.org/mediawiki/2019/b/b4/T--IISc-Bangalore--Ni-NTA_purification.png)

   Fig.3-Ni-NTA protein purification assay

**## Experiments related to ccdB gene system:**

1. **Growth assay:**

   Our ccdB gene is under the promoter araBAD. As the name suggests, this promoter is induced by arabinose. To determine the effect of different quantities of the inducer (arabinose) on the amount of gene product, a growth assay was performed. Our gene product being a toxin affects cell growth. Glucose acts as a repressor for araBAD. In order to determine any leaky transcription, we grew the bacteria with the gene system incorporated in the absence of arabinose and in the presence of different concentrations of glucose. Then we grew the bacteria with different concentrations of the inducer arabinose. We then counted the number of CFU and were able to determine the effect of different concentrations of inducer arabinose on the cells and hence could have an estimate of the comparative amount of the toxin produced.The results for this can be found on the [Result page](https://2019.igem.org/Team%3AIISc-Bangalore/Results).

2. **Protein extraction and purification:**

   To procure the pure CcdB L83S protein so as to conduct further studies on it, we carried out a protein purification procedure. We extracted the protein using the **Column chromatography** method with a CcdA column chromatogram. To confirm whether we were able to extract the protein of interest (CcdB L83S) and have a rough estimate of the purity of the extracted protein, we performed an SDS-PAGE with different elution samples from the column, total cell lysate and the concentrated protein. The results for this can be found on the [Result page](https://2019.igem.org/Team%3AIISc-Bangalore/Results).

   [![Protein extraction-purification assay](https://static.igem.org/mediawiki/2019/b/be/T--IISc-Bangalore--Protein_purification_FINAL1.png)](https://static.igem.org/mediawiki/2019/b/be/T--IISc-Bangalore--Protein_purification_FINAL1.png)

   Fig.4-Protein extraction-purification assay
3. **Protein Thermal Shift assay:**

   Our protein CcdB L83S is a mutant of the wild type CcdB protein. Thus, it’s stability is expected to be different from that of the wild type protein. The melting point of a protein gives us insight into the stability of that protein. Hence, by determining the melting point of CcdB L83S and comparing it to the wild type protein, we can have a qualitative inference of the stability of our protein. To do this, we determined the melting point of our protein by performing a Protein Thermal Shift assay. The results for this can be found on the [Result page](https://2019.igem.org/Team%3AIISc-Bangalore/Results).

   [![Protein thermal shift assay](https://static.igem.org/mediawiki/2019/2/29/T--IISc-Bangalore--Thermal_assay.png)](https://static.igem.org/mediawiki/2019/2/29/T--IISc-Bangalore--Thermal_assay.png)

   Fig.5-Protein thermal shift assay

**## Potential future experiment:**

Opto-T7 has never known to be incorporated in *B. subtilis*. Thus we plan to characterize the system in *B. subtilis* i.e. we want to determine the optimum conditions for the maximal and most efficient functioning of the gene system. To do this we plan to use spectrometric (OD) analysis. We shall measure the initial OD of the bacterial culture. Then, we shall take this culture into different test tubes (3 mL) and shine different wavelengths of light to each test tube for 1-1.5 hrs. After this, the OD measurement of the culture in different test tubes shall be done. This will help us determine the wavelength of light to be shone on the culture to have the most efficient bacteriostatic effect. We shall also change the exposure time of light and measure the OD after different exposure times to find out the most optimal exposure time.

[![](https://static.igem.org/mediawiki/2019/thumb/1/1d/T--IISc-Bangalore--Fb_g.png/120px-T--IISc-Bangalore--Fb_g.png)](https://www.facebook.com/iiscbangaloreigem/)

[![](https://static.igem.org/mediawiki/2019/thumb/5/54/T--IISc-Bangalore--Insta_g.png/120px-T--IISc-Bangalore--Insta_g.png)](https://instagram.com/igem_iisc?igshid=wckh8r4u03xw)

[![](https://static.igem.org/mediawiki/2019/archive/f/f9/20191021164133%21T--IISc-Bangalore--IISc_fc2.png)](https://www.iisc.ac.in/)

[![](https://static.igem.org/mediawiki/2019/archive/e/e6/20191021163311%21T--IISc-Bangalore--IDT_fc2.png)](https://eu.idtdna.com/pages)

[![](https://static.igem.org/mediawiki/2019/b/bf/T--IISc-Bangalore--DBT_g2.png)
![](https://static.igem.org/mediawiki/2019/d/d5/T--IISc-Bangalore--DBT_fc2.png)](http://dbtindia.gov.in/)

[![](https://static.igem.org/mediawiki/2019/archive/a/a1/20191021163502%21T--IISc-Bangalore--NewEngBL_fc2.png)
![](https://static.igem.org/mediawiki/2019/archive/a/a1/20191021163455%21T--IISc-Bangalore--NewEngBL_fc2.png)](https://international.neb.com/)

[![](https://static.igem.org/mediawiki/2019/archive/6/64/20191021162740%21T--IISc-Bangalore--SnapGene_g2.png)
![](https://static.igem.org/mediawiki/2019/archive/6/68/20191021163106%21T--IISc-Bangalore--SnapGene_fc2.png)](https://www.snapgene.com/)

[![](https://static.igem.org/mediawiki/2019/archive/d/d6/20191021163617%21T--IISc-Bangalore--Twist_fc2.png)
![](https://static.igem.org/mediawiki/2019/archive/d/d6/20191021163611%21T--IISc-Bangalore--Twist_fc2.png)](https://twistbioscience.com/)

[![](https://static.igem.org/mediawiki/2019/archive/9/94/20191021163730%21T--IISc-Bangalore--MathWorks_fc2.png)
![](https://static.igem.org/mediawiki/2019/archive/9/94/20191021163722%21T--IISc-Bangalore--MathWorks_fc2.png)](https://in.mathworks.com/)

[![](https://static.igem.org/mediawiki/2019/archive/b/b3/20191021162839%21T--IISc-Bangalore--GenScript_g2.png)
![](https://static.igem.org/mediawiki/2019/archive/f/f4/20191021163004%21T--IISc-Bangalore--GenScript_fc2.png)](https://www.genscript.com/)

[![](https://static.igem.org/mediawiki/2019/archive/a/a4/20191021162515%21T--IISc-Bangalore--Benchling_g2.png)
![](https://static.igem.org/mediawiki/2019/archive/0/04/20191021162240%21T--IISc-Bangalore--Benchling_fc2.png)](https://www.benchling.com/)

Links

[Address](https://goo.gl/maps/BPKKwKU3hd32)

Contact

Best Viewed on Desktop

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:IISc-Bangalore/Results

[![](https://static.igem.org/mediawiki/2019/0/0c/T--IISc-Bangalore--horizontal-logo.png)](https://2019.igem.org/Team%3AIISc-Bangalore)

* Project

  [Description & Inspiration](https://2019.igem.org/Team%3AIISc-Bangalore/Description)
  [Design](https://2019.igem.org/Team%3AIISc-Bangalore/Design)
  [Experiments](https://2019.igem.org/Team%3AIISc-Bangalore/Experiments)
  [Parts](https://2019.igem.org/Team%3AIISc-Bangalore/Parts)
  [Modelling](https://2019.igem.org/Team%3AIISc-Bangalore/Model)
* Lab

  [Protocols](https://2019.igem.org/Team%3AIISc-Bangalore/Lab/Protocol)
  [Growth Regulation](https://2019.igem.org/Team%3AIISc-Bangalore/Growth_Regulation)
  [Assembly](https://2019.igem.org/Team%3AIISc-Bangalore/Assembly)
  [Characterisation](https://2019.igem.org/Team%3AIISc-Bangalore/Characterisation)
  [Improvement](https://2019.igem.org/Team%3AIISc-Bangalore/Improve)
  [Results](https://2019.igem.org/Team%3AIISc-Bangalore/Results)
  [Lab Notebook](https://2019.igem.org/Team%3AIISc-Bangalore/Notebook)
  [Safety](https://2019.igem.org/Team%3AIISc-Bangalore/Safety)
* [Hardware](https://2019.igem.org/Team%3AIISc-Bangalore/Hardware)

  [Introduction](https://2019.igem.org/Team%3AIISc-Bangalore/Hardware)
  [OptoMatic](https://2019.igem.org/Team%3AIISc-Bangalore/OptoMatic)
  [Impedance Method](https://2019.igem.org/Team%3AIISc-Bangalore/OptoMatic/Impedance)
  [Absorbance Method](https://2019.igem.org/Team%3AIISc-Bangalore/OptoMatic/Absorbance)
  [Zeta Potential Method](https://2019.igem.org/Team%3AIISc-Bangalore/OptoMatic/Zeta)
* [Software](https://2019.igem.org/Team%3AIISc-Bangalore/Software)
* Community

  [Collaborations](https://2019.igem.org/Team%3AIISc-Bangalore/Collaborations)
  [Human Practices](https://2019.igem.org/Team%3AIISc-Bangalore/Human_Practices)
  [Integrated Human Practices](https://2019.igem.org/Team%3AIISc-Bangalore/Integrated_HP)
  [Public Engagement](https://2019.igem.org/Team%3AIISc-Bangalore/Public_Engagement)
* Team

  [Philosophy](https://2019.igem.org/Team%3AIISc-Bangalore/Philosophy)
  [Members](https://2019.igem.org/Team%3AIISc-Bangalore/Team)
  [Attributions](https://2019.igem.org/Team%3AIISc-Bangalore/Attributions)

# Results

###### Quick links

* ECFP Characterisation

* Improvements

* CcaS/R System

* OPTO-T7 System

* ccdB System

* Modelling

## Characterization of BBa\_E0020 (ECFP):

The BioBrick [BBa\_E0020](http://parts.igem.org/Part%3ABBa_E0020) was characterized by using the BioBrick [BBa\_I13600](http://parts.igem.org/Part%3ABBa_I13600) as a generator of the ECFP(Engineered Cyan Fluoroscent Protein) and due to availiability in the 20 D well of the Distribution Kit Plate 2. Further, this part was incorporated in BL21 DE3 for the better expression and folding of the protein and was subjected for protein extraction.

![](https://static.igem.org/mediawiki/2019/d/d1/T--IISc-Bangalore--characterisation-gel2.jpg)

**Fig.1**,SDS PAGE Gel for ECFP Characterization. As can be seen from the marked band in the SDS PAGE lies in between the 25 KDa and 32 KDa. The calculated size of the protein of interest is around 28 KDa which is very close to the theoretical one i.e. 26.9 KDa.

![](https://static.igem.org/mediawiki/2019/d/d3/T--IISc-Bangalore--characterisation-gel1.jpg)

**Fig.2**
SDS PAGE after Ammonium sulfate Purification Assay. As can be seen from, there are intensively bright bands at the expected size i.e. 27 KDa in 55% and 60% (w/V) saturation by ammonium sulfate.

Artificially derived from GFP, the engineered cyan fluorescent protein (ECFP) is commonly used as a fluorescent marker in molecular biology studies due to its photostability, high lifespan and relatively low maturation times. To characterize the fluorophore we first performed a full wavelength scan to obtain the excitation and emission spectra of the protein.

![](https://static.igem.org/mediawiki/parts/6/6c/T--IISc-Bangalore--ECFP_spectra.png)

***Fig (1) : Excitation and Emission Spectra for ECFP***

## Improvement Part i2mcherry (Ile)(BBa\_K3165046) :

The protein was expressed under the T7 promoter in *Escherichia coli* (BL21DE3) with 6xHistag at the N-terminal. The transformed bacteria were incubated at 37oC for over 4 hours. The cells were lysed by sonication and the lysate was collected via centrifugation. The lysate was run on an SDS PAGE which showed two bands. The size of the protein of interest along with the 6xHistag is around 27 kDa. The upper band corresponds to the non-truncated protein while the lower band represents the truncated product.

![](https://static.igem.org/mediawiki/parts/f/f2/T--IISc-Bangalore--i2mCherry_%28Ile%29_SDS.png)

***Fig(1) : SDS PAGE for mCherry and i2mCherry (Ile)***

From the SDS PAGE, we clearly observe two bands when using mCherry, while i2mCherry (Ile) gave one very large spot on the polyacrylamide gel with a trailing smear kind of band. The observed dark band is seen at the desired size (roughly around 27 kDa) confirming that the bands correspond to our protein of interest.
Purification using Ni-NTA with [BBa\_K3165046](http://parts.igem.org/Part%3ABBa_K3165046)
The cell lysate thus obtained was purified using Ni-NTA beads as only the non-truncated protein having 6xHistag can bind to the beads. Ideally, the supernatant after binding should have the truncated protein while the eluted fraction should contain the non-truncated protein. This idealization does not hold true as the binding of Ni-NTA is not perfect.

![](https://static.igem.org/mediawiki/parts/5/59/T--IISc-Bangalore--i2mCherry_%28Ile%29_Ni-NTA.png)

***Fig (2) : SDS PAGE for Ni-NTA Purification of i2mCherry (Ile)***

Fluorescence Analysis
Wavelength scans of the protein lysate were performed to obtain the excitation and emission spectra of the fluorescent protein. The fluorescence data obtained were corrected for the blank (untransformed BL21DE3 protein lysate) and the data so obtained is presented below :

![](https://static.igem.org/mediawiki/parts/6/6f/T--IISc-Bangalore--mCherry_Spectra.png)

***Fig(1) : Excitation and Emission Spectra of mCherry***

![](https://static.igem.org/mediawiki/parts/0/0f/T--IISc-Bangalore--i2mCherry_%28Ile%29_Spectra.png)

***Fig(2) : Excitation and Emission Spectra of i2mCherry (Ile)***

## CcaSR System:

This system consists of two subunits: CcaS-GP2 [(BBa\_K3165039)](http://parts.igem.org/Part%3ABBa_K3165039) and CcaR-Ho1-PcyA [(BBa\_K3165036)](http://parts.igem.org/Part%3ABBa_K3165036).The CcaR-Ho1-PcyA subunit was digested with EcoRI and PstI. Similarly, the plasmid pSB1C3 was digested with the same enzymes. Now, these two parts were ligated together and the ligated DNA was used for the transformation of DH5α strain of *Escherichia coli*. Colonies were randomly chosen from these transformed plates with chloramphenicol resistance. The *figures* mentioned below show the enumerated colonies on which plasmid miniprep was performed. These extracted plasmids were then digested with the same restriction endonucleases in order to check for the correct size of the inserted fragment.

Further, from the images, it can be concluded that colony numbers 6, 7, 8, 9 and 18 had inserted fragments of the correct size. We cross-checked the results by doing the colony PCR on only those colonies as the quality of digest wasn't very good.

![](https://static.igem.org/mediawiki/2019/c/c3/T--IISc-Bangalore--CcaR--digest%289-9-19%29.jpg)

CcaR-Ho1-PcyA screening colonies by digestion from Colony 1 to Colony 9

![](https://static.igem.org/mediawiki/2019/1/17/T--IISc-Bangalore--CcaR--digest%2811-9-19%29.jpg)

CcaR-Ho1-PcyA screening colonies by digestion from Colony 1' to Colony 8'

![](https://static.igem.org/mediawiki/2019/7/75/T--IISc-Bangalore--CcaR--digest%2810-9-19%29.jpg)

CcaR-Ho1-PcyA screening colonies by digestion from Colony 1'' to Colony 6''

![](https://static.igem.org/mediawiki/2019/e/e1/T--IISc-Bangalore--CcaR--digest%287%2C10%2C18%29%2810-9-19%29.jpg)

CcaR-Ho1-PcyA screening colonies by digestion from Colony 7'' to Colony 10'' and Colony 18''

![](https://static.igem.org/mediawiki/2019/c/c3/T--IISc-Bangalore--CcaR--digest%289%2C10%2C18%29%2811-9-19%29.jpg)

CcaR-Ho1-PcyA screening colonies by digestion of Colony 9'', Colony 10'' and Colony 18''

**## OPTO-T7 System:**

This system consists of two subunits: T7-NMAG [(BBa\_K3165050)](http://parts.igem.org/Part%3ABBa_K3165050) and PMAG-Gp2 [(BBa\_K3165052)](http://parts.igem.org/Part%3ABBa_K3165052). We PCR amplified the PMAG-GP2 fragment (as shown in the figure below):

![](https://static.igem.org/mediawiki/2019/3/30/T--IISc-Bangalore--synshine-opto_t7_PCR.jpg)

PCR for PMAG-GP2 [(BBa\_K3165052)](http://parts.igem.org/Part%3ABBa_K3165052)

After the PCRing of the PMAG-GP2, it is digested with EcoRI and PstI. Similarly, the plasmid pBS1C was digested with the same enzymes. Now, these two parts were ligated together and the ligated DNA was used for the transformation of DH5α strain of *Escherichia coli*.
Colonies were randomly chosen from these transformed plates with ampicillin and spectinomycin resistance.

Colonies were chosen from these plates and plasmid miniprep was performed. The extracted plasmid was digested with EcoRI restriction enzyme to check for the right size of the linearized ligated plasmid.

![](https://static.igem.org/mediawiki/2019/5/5e/T--IISc-Bangalore--opto-t7-plasmid-1.jpg)

Ligated pBS1C and pBS4S plasmid digested with EcoRI

![](https://static.igem.org/mediawiki/2019/1/15/T--IISc-Bangalore--opto-t7-plasmid-2.jpg)

The plasmids pBS1C and pBS4S digested to obtain linearized backbones

**## CcdAB Antitoxin-Toxin System**

### **Expression and Characterisation**

This part was incorporated in Top10 G (gryA) R462C which is one of the strains that can be used to produce CcdB L83S because of the mutation in Gyrase A making the cells tolerant to it.
The secondary culture was induced with L-Arabinose and a total cell lysate was made by following the protein extraction protocol. Further, an uninduced sample was also separated as a control to check leaky transcription.
The supernatant is subjected to protein purification by incubation in the CcdA column and later eluted to get 10 different samples that are loaded in the SDS PAGE mentioned.

![](https://static.igem.org/mediawiki/parts/4/4b/T--IISc-Bangalore--ccdB_gel1.png)

***Fig.1** - SDS PAGE Gel 1 showing the elutes for the purified **CcdB L83S** mutant protein.*

![](https://static.igem.org/mediawiki/parts/b/b5/T--IISc-Bangalore--ccdB_gel2.jpg)

***Fig.2** - SDS PAGE Gel 2 showing the elutes for the purified **CcdB L83S** mutant protein.*

It can be seen from the SDS PAGE image that there is a very faint band in the well containing the uninduced sample. So, we concluded a very low level of leaky transcription which further can be reduced by the addition of glucose to the culture as glucose acts as a repressor.
Also, the faint band in the pellet section can be used to evaluate the quality of the protein extraction.
As quite evident from the gel, the size of the protein is 11.7 kDa which matches the theoretical value.

### **Determination of the melting point of the CcdB L83S Protein**

We also determined the melting point of the CcdB L83S mutant by performing Thermal Shift Assay.

![](https://static.igem.org/mediawiki/parts/2/2f/T--IISc-Bangalore--ccdB_melting_total.png)

***Fig.3** The full Thermal Shift Assay graph for determining the melting point of **CcdB L83S** mutant protein.*

![](https://static.igem.org/mediawiki/parts/1/15/T--IISc-Bangalore--ccdB_mean_partial_fluore.png)

***Fig.4** The zoomed in picture of the plot as mention in "Fig.3" Thermal Assay graph for determining the melting point of **CcdB L83S** mutant protein.*

As can be seen from Fig.3 and Fig.4, the melting point of the **CcdB L83S** mutant protein is calculated to be 43.03oC. When compared to the melting point of the wild type CcdB protein i.e. 63.8oC, the CcdB L83S protein has a much lower melting temperature. Hence, we concluded that the core mutation resulted in the instability of the CcdB L83S mutant.
In addition to this, Growth Assay can be used on strains like Top10 PJAT to draw the same conclusion i.e. the mutant CcdB L83S is less stable than it's wild type. So, this mutant can be used in regulating the bacterial population to an extent.

### **Growth Assay of CcdB L83S Protein mutant**

To show that CcdB L83S is not as potent as the wild type protein, Top 10 pJET strain of *E. coli* containing the CcdB L83S gene was grown on LB agar plates with gentamicin and ampicillin resistance and containing various concentrations of arabinose and glucose.

![](https://static.igem.org/mediawiki/parts/1/19/T--IISc-Bangalore--zero.jpg)

***Fig.5** - The plate shows the colonies of pJAT strain grown with no amount of arabinose or glucose.*

We can see in **Fig.5** that when LB agar plate with no inducer or repressor was used, colonies of bacteria were obtained, which validated the hypothesis that the CcdB L83S mutant can be grown in strains of *E. coli* which have no GyraseA subunit mutation.

![](https://static.igem.org/mediawiki/parts/8/8e/T--IISc-Bangalore--induction_with_glucose.png)

***Fig.6** - The plates shown have a gradually decreasing concentration of glucose towards the left. A trend can be seen that the colony size increases from left to right.*

As can be seen from **Fig.6**, in the presence of glucose, the bacteria grew with at its normal rate and no effects were seen. It can further be noticed that the spots were more prominent at higher concentrations of glucose, which is valid as glucose is a repressor and it reduces the leaky transcription of the CcdB L83S protein.

![](https://static.igem.org/mediawiki/parts/8/8a/T--IISc-Bangalore--arabinose_plates.png)

***Fig.7** - The plates shown have a gradually increasing concentration of arabinose towards the right. A trend can be seen that the colony size idecreases from left to right.*

From **Fig.7**, we can see that despite the presence of arabinose in plates, we were able to visualize very small-sized colonies. But, at higher concentrations of arabinose, these colonies weren't able to grow.
This shows that CcdB L83S can function as a bacteriostatic protein or a bactericidal toxin under the right conditions, and can be used in strains of *E. coli* not containing mutations in Gyrase A subunit.

**## Modelling: Finding the Michaelis-Menten Constant for E. Coli and B. Subtilus in LB**

The protocol of this experiment can be found [here](https://static.igem.org/mediawiki/2019/1/18/T--IISc-Bangalore--MichalisMenten.pdf).

### Experimental results for *Bacillus subtilis*

![](https://static.igem.org/mediawiki/2019/4/4a/T--IISc-Bangalore--BS05.png)

![](https://static.igem.org/mediawiki/2019/c/c9/T--IISc-Bangalore--BS10.png)

![](https://static.igem.org/mediawiki/2019/0/04/T--IISc-Bangalore--BS25.png)

![](https://static.igem.org/mediawiki/2019/9/92/T--IISc-Bangalore--BS50.png)

![](https://static.igem.org/mediawiki/2019/9/9b/T--IISc-Bangalore--BSMM.png)

From the above data (graphs) it can be seen that the Michaelis-Menten constant for *Bacillus subtilis* is 0.032191 +/- 0.0086875 conc. units.

### Experimental results for *Escherichia coli*

![](https://static.igem.org/mediawiki/2019/8/82/T--IISc-Bangalore--EC05.png)

![](https://static.igem.org/mediawiki/2019/7/7b/T--IISc-Bangalore--EC10.png)

![](https://static.igem.org/mediawiki/2019/3/3a/T--IISc-Bangalore--EC25.png)

![](https://static.igem.org/mediawiki/2019/1/1c/T--IISc-Bangalore--EC50.png)

![](https://static.igem.org/mediawiki/2019/c/c3/T--IISc-Bangalore--EC100.png)

![](https://static.igem.org/mediawiki/2019/6/6c/T--IISc-Bangalore--ECMM.png)

From the above data (graphs) it can be seen that the Michaelis-Menten constant for *Escherichia coli* is 0.01827 +/- 0.01318 conc. units.