iGEM 2018 — Team IISc-Bangalore

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# Team:IISc-Bangalore/Notebook

[![](https://static.igem.org/mediawiki/2018/6/63/T--IISc-Bangalore--phageshift_home.gif)](https://2018.igem.org/Team%3AIISc-Bangalore)

* [Home](https://2018.igem.org/Team%3AIISc-Bangalore)
* Project

  [Overview](https://2018.igem.org/Team%3AIISc-Bangalore/Description)
  [PACMAN](https://2018.igem.org/Team%3AIISc-Bangalore/PACMAN)
  [PAIR](https://2018.igem.org/Team%3AIISc-Bangalore/PAIR)
  [APES](https://2018.igem.org/Team%3AIISc-Bangalore/APES)
  [Design](https://2018.igem.org/Team%3AIISc-Bangalore/Design)
  [Experiments](https://2018.igem.org/Team%3AIISc-Bangalore/Experiments)
  [Parts](https://2018.igem.org/Team%3AIISc-Bangalore/Parts)
  [Model](https://2018.igem.org/Team%3AIISc-Bangalore/Model)
* Lab

  [Notebook](https://2018.igem.org/Team%3AIISc-Bangalore/Notebook)
  [Protocols](https://2018.igem.org/Team%3AIISc-Bangalore/Protocols)
  [Assembly](https://2018.igem.org/Team%3AIISc-Bangalore/Assembly)
  [Results](https://2018.igem.org/Team%3AIISc-Bangalore/Results)
  [InterLab](https://2018.igem.org/Team%3AIISc-Bangalore/InterLab)
  [Improve](https://2018.igem.org/Team%3AIISc-Bangalore/Improve)
* Software

  [PhageModifier](https://2018.igem.org/Team%3AIISc-Bangalore/Software)
* Community

  [Collaborations](https://2018.igem.org/Team%3AIISc-Bangalore/Collaborations)
  [Human Practices](https://2018.igem.org/Team%3AIISc-Bangalore/Human_Practices)
  [Integrated HP](https://2018.igem.org/Team%3AIISc-Bangalore/Integrated_HP)
  [Public Engagement](https://2018.igem.org/Team%3AIISc-Bangalore/Public_Engagement)
  [Safety](https://2018.igem.org/Team%3AIISc-Bangalore/Safety)
  [Survey](https://2018.igem.org/Team%3AIISc-Bangalore/Survey)
* Team

  [Members](https://2018.igem.org/Team%3AIISc-Bangalore/Team)
  [Philosophy](https://2018.igem.org/Team%3AIISc-Bangalore/Philosophy)
  [Attributions](https://2018.igem.org/Team%3AIISc-Bangalore/Attributions)

# Notebook

The following page contains a weekly account of the team's activities in the lab.

Week 1
Basic buffers necessary for the summer session were prepared. Plates, tips and other equipment were autoclaved and kept ready, media prepared.

Week 2
DH5-α comp cells were prepared, and Biobricks BBa\_K16530037, BBa\_K731721, BBa\_K525998, BBa\_J18932, BBa\_K1321337 were transformed into these. (transformation successful)

Week 3
Miniprep of transformed colonies, and digested plasmids were run on a gel. (positive bands)

Week 4
PCR products of 7 Biobricks with VF2/VR primers were run on a gel. (Bands of appropriate sizes were obtained)

Week 5
Assembly method 3 for imCherry was initiated- digest and extraction of T7 adapter backbone, ordered oligo, and BBa\_J18932 with appropriate enzymes for three way ligation.

Week 6
Assembly continued- above fragments were ligated, transformed into DH5-α, transformants checked with colony PCR. Positive colonies were miniprepped, and transformed into E.coli BL21 (DE3).

Week 7
Colony PCR for BL21 (DE3) transformants performed. Protein extraction and purification, followed by SDS-PAGE, Ni-NTA column purification, and fluorescence characterisation for mCherry and imCherry were attempted- no protein obtained in case of imCherry.

Week 8
Phage resistant bacteria were identified for usage in the APES assembly and coevolution model. imCherry assembly was restarted. A part of interlab was done and reported. Phages were also isolated from culture and a new lysate was made, using which different methods of mutations were tried out for APES.

Week 9
Few more iterations of Interlab were carried out. imCherry assembly continued. Mutagenesis for APES and subsequent experiments were continued as well, despite unsuccessful attempts.

Week 10
Inter-lab work (CFU counting) was carried out. Extraction and amplification of the T7 adapter was done. imCherry assembly with T7 adapter was continued. Plaque assay performed, phage lysate obtained.

Week 11
Few more iterations of CFU counting carried out for inter-lab. Ordered DNA arrives. Mutation of phages with different mutagens for APES attempted.

Week 12
Ordered DNA was PCRed, digested, and ligated into digested linearised pSB1C3 backbone, and transformed into DH5-α . Transformants were tested with colony PCR, (negative results obtained) following which the above procedure was repeated. Interlab trials were repeated.

Week 13
Following negative results in second assembly trial, mcp-1 under T7 expression was reassembled. mCherry characterisation was initiated. Interlab trials continue.

Week 14
Mcp-1 under T7 expression, mcp-1+ HlyA and mcp-1+PelB assembly trials repeated. Colony PCR yielded positive results for mcp-1 under T7 colonies. Positive colonies were miniprepped, and digested, and bands of correct size were observed. mCherry was expressed, extracted, and SDS PAGE run. Ni-NTA column purification was done for mCherry, along with complete fluorescence analysis. Repeat of mcp-1+HlyA (under T4 recombination cassette), and mcp-1+PelB assembly.

Week 15
Transformation of the mcp-1 under T7 plasmid into BL21(DE3), and protein expression was done, followed by SDS-PAGE(unsatisfactory results were obtained). Repeat of imCherry assembly procedures initiated with the ordered fragment (Assembly method 1).

Week 16
Assembly procedures (verification of transformed plasmids with colony PCR, and EcoRI+PstI digest following miniprep) of imCherry continued, and positive results were obtained. Assembly of endolysin under constitutive promoter was begun, and positive results were observed after final E+P digest. mcp-1 under T7 expression was electroporated into BL21 (DE3), and following successful electroporation, protein expression was initiated.

Week 17
The process of assembling and preparing the various DNA fragments and making proper usable plasmids continued. (525998, 731721, MCP-1 + HlyA, MCP-1 + PelB). MCP-1 under T-7, whose plasmids were ready, was sent for sequencing. A successful sequencing was followed by attempts to extract the protein. Competent cells weren’t working, so new ones were made.

Week 18
The new competent cells appeared to work (at least DH5-α). The IDT supplied PelB + mcp-1 was tried upon for plasmid creation. The previous assemblies continued. Protein extraction for mcp-1 T7 was tried again and again with different conditions, but proper results not found. imCherry was ligated into an Ampicillin backbone.

Week 19
Previous assemblies continued. λ Red BioBrick assembly initiated. Tried optimisation techniques for MCP-1 T7. imCherry purification, and 6X-His purification initiated, and Endolysin protein expression work begun, following correct sequencing results. Plasmids (for gp37, gp38 and gp57) obtained from Dr Raiij.

Week 20
imCherry extraction and 6X-His purification continued. Previous assemblies continued. SDS-PAGE for mcp-1 along with endolysin run.

Week 21
Continued the previous assemblies. Continued working on gp57/gp38/gp37. mcp-1 extraction continued. Lytic activity characterization for Endolysin was initiated. Phage lysate extraction with BL21(DE3) and MG1655 were carried out.

Week 22
Previous assemblies were continued. Continued assembly of gp37 M-3 and M-4. Extraction of mcp-1 T7 continued. λ Red BioBrick assembly completed, and verified by a digest of the miniprepped plasmid.

Week 23
Work on mcp-1 T7 with Lambda red assembly continued. Meanwhile phage DNA extraction was also done. Electroporation for transformation of DH5α done. SDS PAGE yielded vague results, inclusion bodies suspected for this. Work on gp37 started, where two modifications M1 and M2 were looked into.

Week 24
Endolysin characterisation was done this week. Work on mcp-1 T7 was continued. Electroporation for imCherry was also done. A protocol for removal of inclusion bodies was also undertaken and gp37 work continued with modifications M1,M3 and M4.

Week 25
Dialysis of gp37 suspected to be in inclusion bodies and its treatment with urea was carried out. Folding assay(CD) was performed to verify proper folding. Triple transformations using the plasmids obtained from Dr Raiij was attempted. Assembly of mcp-1+HlyA/PelB under T7 initiated.

Week 26
Assembly of mcp-1+HlyA/PelB under T7 repeated on obtaining negative result. Triple transformation repeated. Assembly of gp37 M1,M3, M4 attempted.

Week 27
Assembly of gp37 M1, M3, M4 repeated. Triple transformation remains unsuccessful. Successful assembly of mcp-1 PelB under T7 expression system verified by E+P digest of miniprepped plasmid. Final mCherry-imCherry truncation profiles done.

Week 28
Successful assembly of mcp-1+HlyA under T7 expression system confirmed by correct sizes E+P digest of miniprepped DNA on gel.

To view our detailed Lab Notebook with daily updates, please download the attached PDF

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

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[![](https://static.igem.org/mediawiki/2018/5/51/T--IISc-Bangalore--IDT_logo.png)](https://eu.idtdna.com/pages)

[![](https://static.igem.org/mediawiki/2018/4/48/T--IISc-Bangalore--NEB_logo.png)](https://www.neb.com/)

[![](https://static.igem.org/mediawiki/2018/a/a0/T--IISc-Bangalore--ODAA_logo.png)](http://www.odaa.iisc.ac.in/)

[![](https://static.igem.org/mediawiki/2018/5/55/T--IISc-Bangalore--Snaogene_logo.png)](https://www.snapgene.com/)

[![](https://static.igem.org/mediawiki/2018/5/58/T--IISc-Bangalore--Mathworks_logo.png)](https://in.mathworks.com/)

Links

* [IISc Bangalore](http://www.iisc.ac.in)
* [IISc UG Program](https://iisc.ac.in/ug/)
* [Complete list of references](https://2018.igem.org/Team%3AIISc-Bangalore/References)

[Address

* Undergraduate department,
* Indian Institute of Science,
* Bangalore - 560012](https://goo.gl/maps/BPKKwKU3hd32)

Contact

* Bhaskar Kumawat
* Team Lead
* kbhaskar@iisc.ac.in

This website is best viewed on a 16:9 aspect ratio screen on the latest version of Google Chrome


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:IISc-Bangalore/Experiments

[![](https://static.igem.org/mediawiki/2018/6/63/T--IISc-Bangalore--phageshift_home.gif)](https://2018.igem.org/Team%3AIISc-Bangalore)

* [Home](https://2018.igem.org/Team%3AIISc-Bangalore)
* Project

  [Overview](https://2018.igem.org/Team%3AIISc-Bangalore/Description)
  [PACMAN](https://2018.igem.org/Team%3AIISc-Bangalore/PACMAN)
  [PAIR](https://2018.igem.org/Team%3AIISc-Bangalore/PAIR)
  [APES](https://2018.igem.org/Team%3AIISc-Bangalore/APES)
  [Design](https://2018.igem.org/Team%3AIISc-Bangalore/Design)
  [Experiments](https://2018.igem.org/Team%3AIISc-Bangalore/Experiments)
  [Parts](https://2018.igem.org/Team%3AIISc-Bangalore/Parts)
  [Model](https://2018.igem.org/Team%3AIISc-Bangalore/Model)
* Lab

  [Notebook](https://2018.igem.org/Team%3AIISc-Bangalore/Notebook)
  [Protocols](https://2018.igem.org/Team%3AIISc-Bangalore/Protocols)
  [Assembly](https://2018.igem.org/Team%3AIISc-Bangalore/Assembly)
  [Results](https://2018.igem.org/Team%3AIISc-Bangalore/Results)
  [InterLab](https://2018.igem.org/Team%3AIISc-Bangalore/InterLab)
  [Improve](https://2018.igem.org/Team%3AIISc-Bangalore/Improve)
* Software

  [PhageModifier](https://2018.igem.org/Team%3AIISc-Bangalore/Software)
* Community

  [Collaborations](https://2018.igem.org/Team%3AIISc-Bangalore/Collaborations)
  [Human Practices](https://2018.igem.org/Team%3AIISc-Bangalore/Human_Practices)
  [Integrated HP](https://2018.igem.org/Team%3AIISc-Bangalore/Integrated_HP)
  [Public Engagement](https://2018.igem.org/Team%3AIISc-Bangalore/Public_Engagement)
  [Safety](https://2018.igem.org/Team%3AIISc-Bangalore/Safety)
  [Survey](https://2018.igem.org/Team%3AIISc-Bangalore/Survey)
* Team

  [Members](https://2018.igem.org/Team%3AIISc-Bangalore/Team)
  [Philosophy](https://2018.igem.org/Team%3AIISc-Bangalore/Philosophy)
  [Attributions](https://2018.igem.org/Team%3AIISc-Bangalore/Attributions)

# **Experiments**

* PACMAN

* Binding Affinity Assay

* PAIR

* Secretion Assay
* Chemotaxis Assay
* Lambda Red recombination

* APES

* Phage MIC Calculation
* UV Mutagenesis
* EMS Mutagenesis

* mCherry Improvement

* Truncation assay

The following is a list of experiments we did to demonstrate the working of one or more parts of our project. Results for the same can be found on the [Results](https://2018.igem.org/Team%3AIISc-Bangalore/Results) page.

## **PACMAN**

### **Binding Affinity Assay**

A direct experimental assay for binding of gp37 to pEtN (phosphoethanolamine) would require a technique like ITC which asks for extremely large concentrations of protein for every run. As such, we decided to take a more structural approach to the assay by using Circular Dichroism measurements as our peek into what is going on at a molecular level. The binding of the ligand will change the structure of the protein giving a minor deviation from the normal spectrum. In the presence of a strongly binding ligand, the spectrum would be shifted further. The difference of the CD spectrum with and without the ligand can thus allow us to qualitatively check its binding affinity for the protein.

![](https://static.igem.org/mediawiki/2018/7/7f/T--IISc-Bangalore--ExpCD1.png)

![](https://static.igem.org/mediawiki/2018/e/e6/T--IISc-Bangalore--ExpCD2.png)

---

## **PAIR**

### **Secretion Assay**

A simple secretion assay for MCP-1 attached to the signal peptides was done to check the secretion efficiencies of both HlyA and PelB. The supernatant obtained after centrifugation of overexpressed flask culture was run on an SDS-PAGE gel (along with the cell pellet) to see if the protein was secreted out successfully.

The band size obtained in the supernatant can be used to determine whether the protein was cleaved after secretion or not.

### **Chemotaxis Assay**

To check whether our recombinant Mcp-1 with and without the signal peptides is active, we used a chemotaxis experiment with the protein purified from *E. coli*. To get rid of LPS contamination the protein was washed multiple times when bound to the column and was dialysed against a sterile buffer.

For Mcp-1 specifically, a series of wash buffers containing decreasing concentrations of urea were used to dialyse the protein sample and refold it from an initial urea concentration of 8M. For Mcp-1, 8M urea solubilization needed to be done because of the tendency of Mcp-1 to form [inclusion bodies](http://parts.igem.org/Part%3ABBa_K2609007) when expressed in *E. coli*.

For the actual assay, we used Ibidi's µ-Slide designed for chemotaxis experiments. The slides were filled with the blank buffer on one side and buffer containing 75ng/ml of the protein on the other (final concentration). Following this, weakly adherent THP-1 cells were loaded in the middle of the slide and their movement was tracked over a span of 12 hrs.

![](https://static.igem.org/mediawiki/2018/4/43/T--IISc-Bangalore--ExoChemo.png)

### **Lambda Red recombination**

The Lambda red recombination system is a combination of three enzymes (recombinases) isolated from the Lambda phage that together increase the basal DNA recombination efficiency in the cell by around four orders of magnitude. These recombinases - called Beta, Exo and Gam - can be expressed from a plasmid and ensure efficient recombination of a dsDNA carrier inside the cell to its homologous regions in either the bacterial genome or another target strand inside the cell. We added a T7 promoter to ZJU-China's 2014 Part [BBa\_K1433005](http://parts.igem.org/Part%3ABBa_K1433005) to convert it into a Lambda red generator ([BBa\_K2609026](http://parts.igem.org/Part%3ABBa_K2609026)).

For PAIR, the Mcp-1 CDS fused to a signal peptide was to be recombined into the T4 genome in place of the native endolysin gene with the use of 50bp homology on both sides of the construct and in the presence of the lambda red proteins. We were unable to do so because of our inability to electroporate the ~169 kbp T4 genome in multiple trials.

![](https://static.igem.org/mediawiki/2018/0/00/T--IISc-Bangalore--ExpLambRed.png)

---

## **APES**

### **Phage MIC Calculation**

One of the few overlooked problems during the administration of phages for therapeutics is the decision of the dosage that a person will require to treat the infection successfully. We felt that a simple correspondence between antibiotics and phages would prove to be extremely useful in this case.

Using multiple dilutions of both phages and antibiotics, we quantified the percentage survival of bacteria over a wide range of concentrations and created curves that could effectively be used to choose the amount of phage that should be administered to create the same effect as a corresponding amount of antibiotic. Different initial concentrations of cells were incubated with varying concentrations of the agent and the percentage survival after 2 hrs was quantified using a resazurin-based viability dye.

![](https://static.igem.org/mediawiki/2018/1/15/T--IISc-Bangalore--ExpMIC.png)

### **UV Mutagenesis**

Phage mutagenesis was induced by exposing phages in a suspension to UV (298nm) for varying amounts of time before propagating them in a susceptible host. The particles obtained after the first propagation were purified from the whole cell lysate and a part of this was used for further mutagenesis. The other part was utilized for a plaque assay to check the viability of phages obtained at each step of mutagenesis and to check their efficacy on resistant bacteria.

### **EMS Mutagenesis**

For chemical mutagenesis, phage particles were incubated with susceptible cells (OD600 0.6, 104 pfu/mL) for 20 mins followed by addition of Ethyl methanesulfonate (EMS). After propogation for 2 hrs, the phages were purified and propogated in the susceptible strain (to ensure amplification of particles that can infect the susceptible strain). They were then extracted from the lysate and were either used for further mutagenesis or to check the susceptibiity of T4 resistant bacteria.

---

## **mCherry Improvement**

### **Truncation assay**

To quantify the truncation from the internal RBS in imCherry (and mCherry), we used a combination of two strategies. 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.

To get a higher order estimate on the truncation, 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 i/mcherry is 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 truncated protein from each fraction was then summed up and was divided by the fluorescence of the non-truncated part to get the relative fractions of the two varieties.

![](https://static.igem.org/mediawiki/2018/1/12/T--IISc-Bangalore--improve_protocol.png)

The results for the improvement can be found on the [Improve](https://2018.igem.org/Team%3AIISc-Bangalore/Improve) page.

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[![](https://static.igem.org/mediawiki/2018/5/51/T--IISc-Bangalore--IDT_logo.png)](https://eu.idtdna.com/pages)

[![](https://static.igem.org/mediawiki/2018/4/48/T--IISc-Bangalore--NEB_logo.png)](https://www.neb.com/)

[![](https://static.igem.org/mediawiki/2018/a/a0/T--IISc-Bangalore--ODAA_logo.png)](http://www.odaa.iisc.ac.in/)

[![](https://static.igem.org/mediawiki/2018/5/55/T--IISc-Bangalore--Snaogene_logo.png)](https://www.snapgene.com/)

[![](https://static.igem.org/mediawiki/2018/5/58/T--IISc-Bangalore--Mathworks_logo.png)](https://in.mathworks.com/)

Links

* [IISc Bangalore](http://www.iisc.ac.in)
* [IISc UG Program](https://iisc.ac.in/ug/)
* [Complete list of references](https://2018.igem.org/Team%3AIISc-Bangalore/References)

[Address

* Undergraduate department,
* Indian Institute of Science,
* Bangalore - 560012](https://goo.gl/maps/BPKKwKU3hd32)

Contact

* Bhaskar Kumawat
* Team Lead
* kbhaskar@iisc.ac.in

This website is best viewed on a 16:9 aspect ratio screen on the latest version of Google Chrome

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:IISc-Bangalore/Results

[![](https://static.igem.org/mediawiki/2018/6/63/T--IISc-Bangalore--phageshift_home.gif)](https://2018.igem.org/Team%3AIISc-Bangalore)

* [Home](https://2018.igem.org/Team%3AIISc-Bangalore)
* Project

  [Overview](https://2018.igem.org/Team%3AIISc-Bangalore/Description)
  [PACMAN](https://2018.igem.org/Team%3AIISc-Bangalore/PACMAN)
  [PAIR](https://2018.igem.org/Team%3AIISc-Bangalore/PAIR)
  [APES](https://2018.igem.org/Team%3AIISc-Bangalore/APES)
  [Design](https://2018.igem.org/Team%3AIISc-Bangalore/Design)
  [Experiments](https://2018.igem.org/Team%3AIISc-Bangalore/Experiments)
  [Parts](https://2018.igem.org/Team%3AIISc-Bangalore/Parts)
  [Model](https://2018.igem.org/Team%3AIISc-Bangalore/Model)
* Lab

  [Notebook](https://2018.igem.org/Team%3AIISc-Bangalore/Notebook)
  [Protocols](https://2018.igem.org/Team%3AIISc-Bangalore/Protocols)
  [Assembly](https://2018.igem.org/Team%3AIISc-Bangalore/Assembly)
  [Results](https://2018.igem.org/Team%3AIISc-Bangalore/Results)
  [InterLab](https://2018.igem.org/Team%3AIISc-Bangalore/InterLab)
  [Improve](https://2018.igem.org/Team%3AIISc-Bangalore/Improve)
* Software

  [PhageModifier](https://2018.igem.org/Team%3AIISc-Bangalore/Software)
* Community

  [Collaborations](https://2018.igem.org/Team%3AIISc-Bangalore/Collaborations)
  [Human Practices](https://2018.igem.org/Team%3AIISc-Bangalore/Human_Practices)
  [Integrated HP](https://2018.igem.org/Team%3AIISc-Bangalore/Integrated_HP)
  [Public Engagement](https://2018.igem.org/Team%3AIISc-Bangalore/Public_Engagement)
  [Safety](https://2018.igem.org/Team%3AIISc-Bangalore/Safety)
  [Survey](https://2018.igem.org/Team%3AIISc-Bangalore/Survey)
* Team

  [Members](https://2018.igem.org/Team%3AIISc-Bangalore/Team)
  [Philosophy](https://2018.igem.org/Team%3AIISc-Bangalore/Philosophy)
  [Attributions](https://2018.igem.org/Team%3AIISc-Bangalore/Attributions)

# Results

* Improvement of mCherry BioBrick

* Assembly of BioBrick Parts

* PAIR

* What Worked
* What Didn't Work

* PACMAN

* What Worked
* What Didn't Work

* APES

The following are the results of all the experiments we performed over the course of the summer.

### Improvement of mCherry BioBrick

Aiming to eliminate the shortcomings of the existing [BBa\_J18932](http://parts.igem.org/Part%3ABBa_J18932) (mCherry RFP), we replaced the internal RBS like sequence, with a generated weaker sequence (with conserved amino acid sequence). As a result, we obtained a BioBrick [BBa\_K2609006](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2609006) that generates mCherry with reduced truncation. The magnitude of truncation is given below:

* **mCherry BBa\_J18932:** 38.8% ± 2.5% Truncation
* **imCherry BBa\_K2609006:** 20.4% ± 2.5% Truncation

Thus, we have produced a BioBrick which has **47.4%** decrease in truncation from the original.
Complete details can be found on the [Improve](https://2018.igem.org/Team%3AIISc-Bangalore/Improve) page.

---

### Assembly of BioBrick Parts

The following parts were assembled, verified by sequencing, and characterised by expression and visualisation on SDS PAGE. These were also submitted to the iGEM Registry to aid teams and labs in their endeavors.

* **BBa\_K2609007 (mcp-1 under T7 expression system)**
  Generates the Mcp-1 chemokine when transformed into a T7 expression strain like E coli. BL21(DE3).
* **BBa\_K2609006 (improved mCherry construct)**
  Generator for improved version of mCherry RFP construct, modified to prevent truncation of protein. Produced mCherry when expressed in a T7 expression strain like E coli. BL21(DE3).
* **BBa\_K2609017 (T4 Endolysin under constitutive promoter)**
  Generates T4 Endolysin (a lysozyme) constitutively.
* **BBa\_K2609026 (Lambda RED recombinases under T7 expression system)**
  Generates the Lambda RED recombination proteins - exo, gam and beta - when transformed into a T7 expression strain like E coli. BL21(DE3).
* **BBa\_K2609013 (gp37(modification 2) under T7 expression system)**
  Generates the tip of the T4 long tail fiber protein when transformed into a T7 expression strain like E coli. BL21(DE3).

The following parts were assembled, verified by sequencing and submitted to the iGEM Registry for the use of other teams and labs.

* **BBa\_K2609008 (mcp-1 + HlyA in T4 recombination cassette) and
  BBa\_K2609009 (mcp-1 + PelB in T4 recombination cassette)**
  Mcp-1 attached to a C terminus HlyA signal peptide with a OmpT linker and N terminus PelB signal peptide respectively, inside a T4 Recombination Cassette. Intended for recombination and replacement of endolysin.
* **BBa\_K2609010 (mcp-1 + HlyA under T7 expression system) and
  BBa\_K2609011 (mcp-1 + PelB under T7 expression system)**
  Generates mcp-1 fused with HlyA and PelB respectively, when transformed into a T7 expression strain like E coli. BL21(DE3). Intended for testing the functioning of the signal peptide HlyA.

The method used to assemble and their verification with all gel images can be found on the [Assembly](https://2018.igem.org/Team%3AIISc-Bangalore/Assembly) page.

---

### PAIR

#### What Worked

**Assembly of DNA Parts**
All the BioBrick parts required for PAIR were assembled and verified to be correct by sequencing. The BioBricks that were assembled are: mcp-1 under T7 expression system (BBa\_K2609007), Lambda RED recombinases under T7 expression system (BBa\_K2609026), and mcp-1 with signal peptides HlyA/PelB under T7 expression system (BBa\_K2609010 & BBa\_K2609011).

![](https://static.igem.org/mediawiki/2018/7/7e/T--IISc-Bangalore--pairgels.png)

**Expression and purification of mcp-1**
Mcp-1 was produced in E coli. BL21(DE3) by using the part *mcp-1 under T7*. Protein was produced by induction at OD600=0.6 with 250μM IPTG and grown for 16 hours at 25°C. The protein was forming inclusion bodies and hence was solubilized in 8M urea. This was directly applied to an Ni-NTA column and purified. The 6xHis tag purified protein was then dialized to get pure mcp-1 in PBS. Performing a Bradford Assay on the dialized protein gave a concentration of 49.8 μg/mL.

![](https://static.igem.org/mediawiki/2018/8/89/T--IISc-Bangalore--mcp1Purification2.png)

**Characterisation of Lambda RED BioBrick**
We characterised the Lambda RED BioBrick BBa\_K1433005 using the generator BBa\_K2609026 by expression and visualising on SDS PAGE. This part is under T7 expression system and hence was transformed into E coli BL21(DE3) and induced with 500 μM IPTG at OD600 = 0.6 and grown for 3 hours at 37°C. Cell pellet was boiled with SDS sample buffer and loaded ran on SDS PAGE.
Bands were observed slightly below 32kDa ladder band and at the 25kDa ladder band. These two correspond to the bet protein (29.7 kDa) and the exo protein (25.9 kDa). The other protein - gam (16.3 kDa) - could not be seen on the gel. This could be because the part has only one RBS sequence for all three proteins. It could be either that the third protein is being produced in very small amounts or not at all.

![](https://static.igem.org/mediawiki/2018/7/7e/T--IISc-Bangalore--lambdaT7exp2.png)

**Characterisation of T4 Endolysin BioBrick**
We characterised the BioBrick BBa\_K2609001 using the generator BBa\_K2609017 by the size of the protein produced and by its function. Since, this part is under a constitutive promoter, it cannot be induced. Hence, bands of E coli BL21(DE3) containing this part were compared with the bands of E coli BL21(DE3). The SDS PAGE is shown below.

![](https://static.igem.org/mediawiki/2018/a/ad/T--IISc-Bangalore--endopage.png)

![](https://static.igem.org/mediawiki/parts/a/a2/Lytic_Activity_Characterisation_of_Endolysin_iGEM18_IISc_Bangalore.jpg)

We also characterised the lystic activity of recombinantly produced T4 Endolysin. For this, we extracted protein from E coli BL21(DE3) containing the endolsyin plasmid along with wild type. B Cereus, a gram positive bacteria, was grown to mid log phase and then suspended in 0.2mM Tris-HCl (pH 8). 0.1 volumes of the following were added:

* Protein extract from E coli BL21(DE3) containing T4 Endolysin
* Protein extract from wild type E coli BL21(DE3)
* Protein lysis buffer
* Control blank buffer

OD600 was measured using a plate reader continuously over a span of two hours. We obtained the graph shown above after taking average from multiple replicates. From this, we see that endolysin reduces B Cereus cell density faster than the other solutions. This is confirmation that recombinantly produced T4 Endolysin is functional and acts as a lysozyme.

**Extraction of Phage DNA**
Using an enzymatic protocol with Proteinase K and DNAse, we obtained T4 bacteriophage genomic DNA. The DNA could be seen precipitating after on addition of isopropanol. Following spectrophotometric analysis of the DNA, it was found to be very high concentration (~2 μg/μL) and high purity (A260/A280 = 1.8).

![](https://static.igem.org/mediawiki/2018/f/f4/T--IISc-Bangalore--phageDNA2.png)

#### What Didn't Work

**Electroporation of T4 phage DNA**
We were unable to electroporate the extremely long ~169kbp genome of T4 bacteriophage. We tried electroporation of the extracted DNA into different strains of E coli in multiple trials with no success. We also consulted the highly experienced in bacteriophage research, Prof. Ryland Young and Prof. Graham Hatfull, for their inputs. Even so, we were not able to troubleshoot the problem.

**Recombination with the Lambda RED System**
We were unable to recombine with using the Lambda RED BioBrick BBa\_K2609026 made from BBa\_K1433005. We tried a test recombination of plasmids in multiple trials but were unable to generate any recombinants.

**Generation of T4 phage recombinants**
The success of this part depended on the success of the previous two parts. Since, neither of them gave any positive result, we were unable to proceed to this step.

---

### PACMAN

#### What Worked

**Expression and Purification of Wild Type gp37**
In order to isolate gp37 for further studies, it was essential that it was properly folded. This protein needs two chaperones for correct folding i.e. gp38 and gp57.
Thus, we did a triple transformation of all the three gene-containing plasmids, expressed by induction with 500μM IPTG at OD600=0.6 and grown at 25°C for 10 hours.
**Folding and Ligand Interaction of Wild Type gp37**
A CD (Circular Dichroism) measurement was performed, and this CD spectrum showed a dip at the wavelength corresponding to exclusively the β pleated structure, hence giving us substantial evidence of its correct folding.
We measured CD Spectra of WT gp37 in the presence of different concentrations of the ligand PEtN. We plotted the minimum angle against ligand concentration and obtained the below graph. From the we can conclude that there is no binding between WT gp37 and PEtN as there is no observable trend. This can be explained by the fact that the thermal energy of the protein and the ligand are greater than the interaction energy between them.

![](https://static.igem.org/mediawiki/2018/9/94/T--IISc-Bangalore--wtgp37pur.png)

![](https://static.igem.org/mediawiki/2018/e/e2/T--IISc-Bangalore--gp37CD2.png)

![](https://static.igem.org/mediawiki/2018/3/32/T--IISc-Bangalore--gp37binding2.png)

**Assembly and expression of gp37 M2 under T7 expression system**
The BioBrick part BBa\_K2609013 was assembled, and verified to be correct through DNA sequencing. It was then transformed into E coli BL21(DE3) and induced with 500μM IPTG at OD600 = 0.6 and grown for 3 hours at 37°C. The cell pellet was then loaded on SDS PAGE.

![](https://static.igem.org/mediawiki/2018/d/d0/T--IISc-Bangalore--gp37m2dig2.png)

![](https://static.igem.org/mediawiki/2018/3/3e/T--IISc-Bangalore--gp37m2page.png)

#### What Didn't Work

**Assembly of gp37 M1,M3 and M4**
The other three modifications of gp37 could not be assembled even after multiple trials.

**Purification of Correctly Folded gp37 M2**
Since gp37 needs its chaperones gp38 & gp57 to fold correctly, the three proteins had to be triple transformed and then expressed. Following multiple trials of triple transformation, we did not obtain a transformant that expressed all three proteins. Thus, we could not obtain correctly folded gp37 M2 to proceed further.

---

### APES

The APES experiments failed to produce reproducibly, across trials, a sufficient number of mutant phages capable of infecting and lysing T4-resistant *E. coli*.

![](https://static.igem.org/mediawiki/2018/4/44/T--IISc-Bangalore--page_Str2T4min5.png)*Plate showing sample 2 of resistant bacteria plated with T4 after irradiation with UV for 5 minutes. No plaques were observed.*

![](https://static.igem.org/mediawiki/2018/c/c4/T--IISc-Bangalore--page_MG1655T4min5.png)*Plate showing wild-type strain MG1655 plated with T4 after irradiation with UV for 5 minutes. Tiny plaques can be observed.*

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Links

* [IISc Bangalore](http://www.iisc.ac.in)
* [IISc UG Program](https://iisc.ac.in/ug/)
* [Complete list of references](https://2018.igem.org/Team%3AIISc-Bangalore/References)

[Address

* Undergraduate department,
* Indian Institute of Science,
* Bangalore - 560012](https://goo.gl/maps/BPKKwKU3hd32)

Contact

* Bhaskar Kumawat
* Team Lead
* kbhaskar@iisc.ac.in

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