iGEM 2019 — Team IISER_Bhopal

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

# Team:IISER Bhopal/Notebook

iGEM IISER Bhopal

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[## E.L.S.A](https://2019.igem.org/Team%3AIISER_Bhopal)

* [Home](https://2019.igem.org/Team%3AIISER_Bhopal)
* Team

  Team Members
  [Collaborations](https://2019.igem.org/Team%3AIISER_Bhopal/Collaborations)
* Project

  [Introduction](https://2019.igem.org/Team%3AIISER_Bhopal/Introduction)
  [Description](https://2019.igem.org/Team%3AIISER_Bhopal/Description)
  [Design](https://2019.igem.org/Team%3AIISER_Bhopal/Design)
  [Modelling](https://2019.igem.org/Team%3AIISER_Bhopal/Model)
  [Protocols](https://2019.igem.org/Team%3AIISER_Bhopal/Experiment)
  Notebook
  [Results](https://2019.igem.org/Team%3AIISER_Bhopal/Results)
  [References](https://2019.igem.org/Team%3AIISER_Bhopal/References)
* Parts

  [Basic Parts](https://2019.igem.org/Team%3AIISER_Bhopal/Basic_Parts)
  [Improved Parts](https://2019.igem.org/Team%3AIISER_Bhopal/Improved_Parts)
* [Safety](https://2019.igem.org/Team%3AIISER_Bhopal/Safety)
* Human Practices

  [Human Practices](https://2019.igem.org/Team%3AIISER_Bhopal/Human_Practices)
  [Integrated Human Practices](https://2019.igem.org/Team%3AIISER_Bhopal/Integrated_Human_Practices)
  [Attributions](https://2019.igem.org/Team%3AIISER_Bhopal/Attributions)
* [Judging Form](https://igem.org/2019_Judging_Form?id=3121)

Notebook

## MAY

[#### First Week of May](single.html)

* Beginning of lab work.
* SOB,SOC, TE, TB, TAE prepared
* LB media and LB agar plates prepared
* Competent cell preparation of E . coli (DH5a) carried out but failed

#### Second Week of May

* Comp cell preparation of E. coli (DH5a) attempted thrice and failed.

#### Third Week of May

* iGEM kit arrived.
* Comp cell preparation of E. coli (DH5a) successful and competency checked.
* PUC-18 plasmid isolation and stock preparation.

#### Fourth Week of May

* AFP, CPN-10, CPN-60 transformation carried out individually.
* Plasmid isolation of the above transformations carried out.

[#### Fifth Week of May](single.html)

* Primer resuspension followed by primer stock preparation carried out.

## JUNE

![responsive image](https://static.igem.org/mediawiki/2019/a/ae/T--IISER_Bhopal--jwk11.png)

##### Gradient PCR-1 for Cpn10

![responsive image](https://static.igem.org/mediawiki/2019/8/8f/T--IISER_Bhopal--jwk12.png)

##### Gradient PCR-1 for AFP

![responsive image](https://static.igem.org/mediawiki/2019/5/52/T--IISER_Bhopal--jwk13.png)

##### Gradient PCR-1 for Cpn60

![responsive image](https://static.igem.org/mediawiki/2019/4/43/T--IISER_Bhopal--jwk14.png)

##### Gradient PCR-2 for Cpn10 PCR-1 product

![responsive image](https://static.igem.org/mediawiki/2019/2/2e/T--IISER_Bhopal--jwk16.png)

##### Amplification of AFP

![responsive image](https://static.igem.org/mediawiki/2019/0/0b/T--IISER_Bhopal--jwk17.jpg)

##### Amplification of Cpn10

![responsive image](https://static.igem.org/mediawiki/2019/2/24/T--IISER_Bhopal--jwk18.jpg)

##### Amplification of Cpn60

[Previous](#carousel-jwk1)
[Next](#carousel-jwk1)

[#### First Week of June](single.html)

* Gradient PCR-1 of AFP, CPN-10 and CPN-60 carried out successfully.
* Gradient PCR-2 using products of PCR-1 carried out successfully for AFP and Cpn-10 but failed for Cpn-60.
* Digestion of AFP, CPN-10( PCR-2 product) carried out successfully.
* Digestion of PUC-18 plasmid carried out but failed.

Details

![responsive image](https://static.igem.org/mediawiki/2019/8/88/T--IISER_Bhopal--jwk21.jpg)

##### SOEing of AFP + Cpn10

[Previous](#carousel-jwk2)
[Next](#carousel-jwk2)

#### Second Week of June

* Gradient PCR-1 and PCR-2 of CPN-60 carried out but failed.
* Digestion of PUC-18 plasmid carried out successfully.
* LB media and LB agar plates prepared(link to protocol)
* SOEing PCR of AFP+CPN-10 successfully carried out.

Details

#### Third Week of June

* Gradient PCR-2 for Cpn-60 carried out but failed.
* IPTG + X-Gal plates prepared.
* Ligation of PUC-18+AFP carried out successfully.
* Ligation of PUC-18+CPN-10 carried out but failed.
* SOEing PCR of AFP-CPN-10+CPN-60 carried out but failed.

![responsive image](https://static.igem.org/mediawiki/2019/1/1c/T--IISER_Bhopal--jwk41.jpg)

##### Linearisation of pUC18 plasmid

![responsive image](https://static.igem.org/mediawiki/2019/4/47/T--IISER_Bhopal--jwk42.jpg)

##### Colony PCR for AFP ligation

![responsive image](https://static.igem.org/mediawiki/2019/0/07/T--IISER_Bhopal--jwk43.png)

##### SOEing of AFP-Cpn10 with lac promoter

![responsive image](https://static.igem.org/mediawiki/2019/9/99/T--IISER_Bhopal--jwk44.jpg)

##### SOEing PCR of the lac promoter, AFP and Cpn10

![responsive image](https://static.igem.org/mediawiki/2019/6/6c/T--IISER_Bhopal--jwk45.jpg)

##### SOEing PCR of AFP and Cpn10

![responsive image](https://static.igem.org/mediawiki/2019/b/b0/T--IISER_Bhopal--jwk46.jpg)

##### Digestion of pUC18 and TF of *P. haloplanktis* and *P. frigidicola*

[Previous](#carousel-jwk4)
[Next](#carousel-jwk4)

#### Fourth Week of June

* Touchdown PCR for SOEing of AFP-CPN-10+CPN-60 carried out but failed.
* Hotstart PCR of CPN-60 using PCR-1 product carried out but failed.
* Vector linearisation of PUC-18 plasmid successfully carried out using PCR.
* Colony PCR of AFP ligation carried out successfully.
* Freeze thaw cycle for AFP was checked but failed.
* SOEing of CPN-60 and PUC-18 backbone carried out but failed.
* Digestion of Trigger Factor part(TF) and PUC-18 conducted successfully.
* SOEing of AFP-CPN-10 and Part A carried out successfully.
* SOEing of AFP-CPN-10-Part A and CPN-60 carried out but failed.

Details

## JULY

![responsive image](https://static.igem.org/mediawiki/2019/d/dc/T--IISER_Bhopal--lwk11.jpg)

##### Cloned Trigger Factor of P. haloplanktis in pUC18

![responsive image](https://static.igem.org/mediawiki/2019/a/af/T--IISER_Bhopal--lwk12.jpg)

#####

![responsive image](https://static.igem.org/mediawiki/2019/f/f8/T--IISER_Bhopal--lwk13.jpg)

##### Digestion of Cpn10 and Cpn60

[Previous](#carousel-lwk1)
[Next](#carousel-lwk1)

[#### First Week of July](single.html)

* Ligation of PUC-18 and TF of P.haloplanktis carried out successfully.
* Ligation of PUC-18 and TF of P.frigidicola carried out but failed.
* Gradient PCR-2 of CPN-60 with new primers carried out but failed.
* SOEing of AFP-CPN-10-Part A and CPN-60 with new primers carried out but failed.
* Digestion of CPN-10 & CPN-60 directly from iGem kit carried out successfully.
* Screening of AFP found in colony PCR carried out but failed.
* Screening to check the cloning of P. haloplanktis successfully carried out.
* Screening to check the cloning of P. frigidicola carried out but failed.

Details

![responsive image](https://static.igem.org/mediawiki/2019/2/20/T--IISER_Bhopal--lwk21.jpg)

##### Ligation of pUC18 plasmid with Cpn10

[Previous](#carousel-lwk2)
[Next](#carousel-lwk2)

#### Second Week of July

* Ligation of CPN-10 in PUC-18 backbone carried out successfully.
* Colony PCR to check the cloning of CPN-10 carried out successfully.
* Characterization of P.haloplanktis TF carried out.

Details

#### Third Week of July

* Colony PCR of P. frigidicola carried out but failed.
* Digestion of Cpn-10 in PUC-18 carried out successfully.
* Plasmid isolation for Cpn-10+PUC-18 carried out.
* PCR carried out to confirm if the plasmids are correct.

#### Fourth Week of July

* Digestion of Cpn-60+psBIC3 carried out successfully.
* Sequential digestion of Cpn-60 in PUC-18 carried out.
* Colony PCR for TF in P. haloplanktis in PUC-18.
* Plasmid isolation for Cpn10 in pUC18.
* And for Cpn60 in pSB1C3.
* Checking plasmids for Cpn10 in pUC18 - by PCR.

![responsive image](https://static.igem.org/mediawiki/2019/0/05/T--IISER_Bhopal--lwk51.jpg)

##### Ligation of pUC18 with Cpn60

![responsive image](https://static.igem.org/mediawiki/2019/a/a7/T--IISER_Bhopal--lwk52.jpg)

##### Colony PCR for pUC18 plasmid fused with Cpn60

[Previous](#carousel-lwk5)
[Next](#carousel-lwk5)

[#### Fifth Week of July](single.html)

* Cloning of Cpn60 in pUC18.
* Digestion sequential (two digestion reactions one after the other - gel extraction of digestions).
* Ligation of the digested Cpn60 and the digested vector.
* Transformation of the ligated product.
* Master plate for the colonies - patching the colonies.
* Optimizing the IPTG concentration - TF 96 well plate.
* Colony PCR - confirming Cpn60 in pUC18 - M13 F and R.

Details

## AUGUST

[#### First Week of August](single.html)

* Characterization of Cpn-60.

[#### Fifth Week of August](single.html)

* Sequencing of PUC-18+Cpn-10+Cpn-60 carried out.

## SEPTEMBER

[#### First Week of September](single.html)

* Amplification of pET 28a carried out but failed.
* Amplification of *P. frigidicola* TF carried out.

#### Second Week of September

* Transformation of iGEM part- stress sensor pSB1C3 carried out in Rosetta and DH5a selection in Rosetta was not successful (reason: Rosetta is inherently resistant to chloramphenicol).
* Plasmid extraction from a transformed DH5a colony was carried out.
* Overlapping PCR carried out to replace Lac promoter with Ptrc promoter at different annealing temperatures(56 ℃, 57 ℃, 58 ℃, 59 ℃).
* From the above PCR amplification, two fragments were extracted(1085bp & 2018bp) and were further amplified.
* Overlap extension PCR with the amplified fragments was carried out.
* Digestion of the products of overlap extension PCR and pET 28a was carried out(with same restriction enzyme).
* Ligation and cloning of digested product was carried out.

## OCTOBER

![responsive image](https://static.igem.org/mediawiki/2019/a/aa/T--IISER_Bhopal--grow1.jpeg)

![responsive image](https://static.igem.org/mediawiki/2019/a/a1/T--IISER_Bhopal--grow2.jpeg)

![responsive image](https://static.igem.org/mediawiki/2019/7/77/T--IISER_Bhopal--grow3.jpeg)

##### Performance Optimization

[Previous](#carousel-owk1)
[Next](#carousel-owk1)

[#### Growth Curves](single.html)

@iGEM IISERB


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

# Team:IISER Bhopal/Experiments

iGEM IISER Bhopal

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[## E.L.S.A](https://2019.igem.org/Team%3AIISER_Bhopal)

* [Home](https://2019.igem.org/Team%3AIISER_Bhopal)
* Team

  Team Members
  [Collaborations](https://2019.igem.org/Team%3AIISER_Bhopal/Collaborations)
* Project

  Introduction
  [Description](https://2019.igem.org/Team%3AIISER_Bhopal/Description)
  Design
  [Modelling](https://2019.igem.org/Team%3AIISER_Bhopal/Model)
  Experiment
  Notebook
  Results
* Parts

  Basic Parts
  Composite parts
  Improved Parts
* Safety
* Human Practices

  [Human Practices](https://2019.igem.org/Team%3AIISER_Bhopal/Human_Practices)
  Education & Engagement
  Integrated Human Practices
  [Attributions](https://2019.igem.org/Team%3AIISER_Bhopal/Attributions)
* [Judging Form](https://igem.org/2019_Judging_Form?id=3121)

#### AAAAAAAAAAAAAAA

Our project this year is aimed at developing a cold-tolerant chassis for the stable expression of psychrophilic genes in mesophilic hosts.

Did you tune out already? Worry not! Sit back, and let our garrulous smooth talkers spin a fascinating yarn to keep you captivated. After all, iGEM’s all about learning the fun way!

Let’s begin with the basics. Mesophilic organisms are your regular next-door microbes which grow optimally in moderate temperatures (usually defined between a fairly nippy 20°C to and a decidedly uncomfortable 45°C). Their more adventurous cousins are the extremophiles - the legends who not only survive but outright thrive, in the face of some of Earth’s harshest environments. Be it corrosive pH ranges, crushing pressures, near-melting temperatures, tons of radiation, or (of particular interest to us) freezing cold conditions, these wily minions have well and truly proven their hardiness.

![](img/app-profile-mockup.png)

![](img/app-profile-mockup.png)

#### Capture every moments and share it to all your friends

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iGEM IISERB

# 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)

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

# Team:IISER Bhopal/Results

iGEM IISER Bhopal

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[## E.L.S.A](https://2019.igem.org/Team%3AIISER_Bhopal)

* [Home](https://2019.igem.org/Team%3AIISER_Bhopal)
* Team

  [Team Members](https://2019.igem.org/Team%3AIISER_Bhopal/Team_Members)
  [Collaborations](https://2019.igem.org/Team%3AIISER_Bhopal/Collaborations)
* Project

  [Introduction](https://2019.igem.org/Team%3AIISER_Bhopal/Introduction)
  [Description](https://2019.igem.org/Team%3AIISER_Bhopal/Description)
  [Design](https://2019.igem.org/Team%3AIISER_Bhopal/Design)
  [Modelling](https://2019.igem.org/Team%3AIISER_Bhopal/Model)
  [Protocols](https://2019.igem.org/Team%3AIISER_Bhopal/Experiment)
  [Notebook](https://2019.igem.org/Team%3AIISER_Bhopal/Notebook)
  [Results](https://2019.igem.org/Team%3AIISER_Bhopal/Results)
  [References](https://2019.igem.org/Team%3AIISER_Bhopal/References)
* Parts

  [Basic Parts](https://2019.igem.org/Team%3AIISER_Bhopal/Basic_Parts)
  [Improved Parts](https://2019.igem.org/Team%3AIISER_Bhopal/Improved_Parts)
* [Safety](https://2019.igem.org/Team%3AIISER_Bhopal/Safety)
* Human Practices

  [Human Practices](https://2019.igem.org/Team%3AIISER_Bhopal/Human_Practices)
  [Integrated Human Practices](https://2019.igem.org/Team%3AIISER_Bhopal/Integrated_Human_Practices)
  [Attributions](https://2019.igem.org/Team%3AIISER_Bhopal/Attributions)
* [Judging Form](https://igem.org/2019_Judging_Form?id=3121)

Results

#### Trigger Factor of *Pseudoalteromonas haloplanktis*

Immediately after synthesizing the TF we had to transform it into the appropriate cells. Once transformed, plasmid extraction was performed and the gel obtained may be seen in Figure 1.1. Post extraction, the plasmid was employed to transform cells, and its effect on cell growth was observed throughout the log phase. Our literature survey and IHP had helped us understand that optimal yield of different proteins is subject to different growth conditions - hence it was necessary for us to perform growth curve analysis and observe the effect of the Trigger Factor's induction on cell growth. The observations obtained for the same are given below. The temperatures chosen were 37°C (as a model for optimal conditions) and 18°C (as a model for suboptimal conditions). While we ideally would have liked to achieve even lower temperatures (as our model clearly predicts that TF could survive near 0°C-like conditions), we were most unfortunately limited by the available infrastructural facilities.

![](https://static.igem.org/mediawiki/2019/e/eb/T--IISER_Bhopal--tfgelimageresult.png)

**Fig.1.1:** Gel image of Trigger Factor *P.haloplanktis* cloned in pUC18.

![](https://static.igem.org/mediawiki/2019/f/f0/T--IISER_Bhopal--tfhaloresultplate.png)

**Fig.1.2:** Trigger Factor *P.haloplanktis* cloned in pUC18.

####

#### Growth Curve at 37°C

![](https://static.igem.org/mediawiki/2019/a/a8/T--IISER_Bhopal--tf37groresult.jpeg)

#### Growth Curve at 18°C

![](https://static.igem.org/mediawiki/2019/7/79/T--IISER_Bhopal--tf18groresult.jpeg)

#### Cpn60

Cpn 60 was successfully digested from the parent backbone pSB1C3, which was confirmed on gel via size confirmation and sequencing exercises. As this part had already been submitted by the 2011 Amsterdam team, we decided to further characterize it (as a part of the Bronze medal criteria) and check how its induction affected cell growth. The results of the growth analysis may be seen in the graphs provided below. The temperatures chosen were 37°C (as a model for optimal conditions) and 18°C (as a model for suboptimal conditions). While we ideally would have liked to achieve even lower temperatures (as our model clearly predicts that TF could survive near 0°C-like conditions), we were most unfortunately limited by the available infrastructural facilities.

![](https://static.igem.org/mediawiki/2019/d/d3/T--IISER_Bhopal--cpn60cloneresult.png)

**Fig.2.1:** Gel Image of Cpn60 cloned in pUC18

![](https://static.igem.org/mediawiki/2019/e/e0/T--IISER_Bhopal--cpn60cloneplaresult.png)

**Fig.2.2:** Cpn60 cloned in pUC18

####

#### Growth Curve at 37°C

![](https://static.igem.org/mediawiki/2019/c/cf/T--IISER_Bhopal--6037groresult.jpeg)

#### Growth Curve at 18°C

![](https://static.igem.org/mediawiki/2019/3/35/T--IISER_Bhopal--6018groresult.jpeg)

#### Cpn10/Cpn60

We obtained the Cpn 10 /60 by sequential cloning of Cpn 60 and Cpn 10 into pUC18 plasmid (in that particular order). Similar to GroEL/GroES, they function by associating with each other so as to enable better folding of proteins. We characterized the composite part and checked how its induction affected cell growth. The results of the growth analysis may be seen in the graphs provided below. The temperatures chosen were 37°C (as a model for optimal conditions) and 18°C (as a model for suboptimal conditions). While we ideally would have liked to achieve even lower temperatures (as our model clearly predicts that TF could survive near 0°C-like conditions), we were most unfortunately limited by the available infrastructural facilities.

![](https://static.igem.org/mediawiki/2019/3/3c/T--IISER_Bhopal--cpn1060result.png)

**Fig.3.1:** Gel Image of Cpn10/60 cloned in pUC18

![](https://static.igem.org/mediawiki/2019/f/f7/T--IISER_Bhopal--cpn1060coleplateresult.png)

**Fig.3.2:** Cpn10/60 cloned in pUC18

####

#### Growth Curve at 37°C

![](https://static.igem.org/mediawiki/2019/1/1f/T--IISER_Bhopal--106037groresult.jpeg)

#### Growth Curve at 18°C

![](https://static.igem.org/mediawiki/2019/0/09/T--IISER_Bhopal--106018groresult.jpeg)

#### Chaperonin Comparitive Analysis

For a wholesome understanding of how different chaperones peak at different temperatures, we set up a 96 well experiment to check the same. A growth curve was plotted for three different temperatures, this data was also used by our modelling team to extrapolate the minimum temperature at which the cells could survive. An SDS page was run for the whole cell lysates of the samples to confirm the presence of their respective proteins.

#### Growth Curve at 37°C

![](https://static.igem.org/mediawiki/2019/2/2c/T--IISER_Bhopal--37compgrocur.jpeg)

#### Growth Curve at 23°C

![](https://static.igem.org/mediawiki/2019/0/01/T--IISER_Bhopal--23compgrocur.jpeg)

#### Growth Curve at 18°C

![](https://static.igem.org/mediawiki/2019/a/a7/T--IISER_Bhopal--18compgrocur.jpeg)

![](https://static.igem.org/mediawiki/2019/0/03/T--IISER_Bhopal--sdslastresult.jpeg)

**Fig.4.1:** Results for SDS-PAGE of whole cell lysates of cell lines containing TF, Cpn60 and empty vector(pUC18) with Rosetta as a control.

@iGEM IISERB