iGEM 2019 — Team IISER_Kolkata

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# Team:IISER Kolkata/Notebook

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* Project

  + [Description](https://2019.igem.org/Team%3AIISER_Kolkata/Description)
  + [Design](https://2019.igem.org/Team%3AIISER_Kolkata/Design)
  + [Experiments](https://2019.igem.org/Team%3AIISER_Kolkata/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AIISER_Kolkata/Notebook)
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  + [Results](https://2019.igem.org/Team%3AIISER_Kolkata/Results)
  + [Model](https://2019.igem.org/Team%3AIISER_Kolkata/Model)
* Parts

  + [Overview](https://2019.igem.org/Team%3AIISER_Kolkata/Parts)
  + [Basic](https://2019.igem.org/Team%3AIISER_Kolkata/Basic_Part)
  + [Composite](https://2019.igem.org/Team%3AIISER_Kolkata/Composite_Part)
  + [Collection](https://2019.igem.org/Team%3AIISER_Kolkata/Part_Collection)
* [Safety](https://2019.igem.org/Team%3AIISER_Kolkata/Safety)
* Humanity

  + [Overview](https://2019.igem.org/Team%3AIISER_Kolkata/Practices_Overview)
  + [Integrated HP](https://2019.igem.org/Team%3AIISER_Kolkata/Human_Practices)
  + [Engagement](https://2019.igem.org/Team%3AIISER_Kolkata/Public_Engagement)
  + [Social](https://2019.igem.org/Team%3AIISER_Kolkata/Social_Studies)

# Notebook

Every project needs organisation and proper planning to succeed. We kept a detailed record of all the activities conducted, for future reference and ease of management. Following is the Notebook section, where we have described our work timeline, beginning from the selection of the team to the Wiki Freeze.

### February

The team was selected on the 8th of February and we had our very first meeting on the following day. In the next few weeks, we went through the pros and cons of each others' iGEM project ideas proposed for the selection process. Finally, after rigorous discussions, we reached a consensus to work on the neglected tropical disease named Leishmaniasis which has a hotspot in the location we are currently residing in. We designed a rough genetic circuit that could sense the change in NO concentration inside the macrophage infected with *Leishmania* parasite and presented the crude idea to Dr. Rupak Datta, a professor working on *Leishmania major*, and gathered relevant insights from him.

### March

iBEC is a project funding initiative taken by the Government of India to provide monetary support to the Indian teams to participate in iGEM. In order to participate in the competition, the team has to send a complete proposal of what they plan to go for the given year of iGEM. Thus we troubleshooted all the potential loopholes in our project proposal and refining it as much as possible. By the 15th of March the proposal was ready and we had sent the hard copy of it to the Department of Biotechnology headquarters in New Delhi.
For the next few weeks, we explored many potential sponsors to fund our project. We spoke to many companies working in the field of synthetic biology for the same.

### April

End Semester Exams!

### May

Head of the Department of Biological Sciences of IISER Kolkata allocated lab space for the team to work in the summer of 2019(May-October).
Collected glass apparatus and reagents from last year’s iGEM team and common lab, and sterilized the apparatus.
Started working on preparing the list of reagents, enzymes, new apparatus, consumables, cell culture requirements, etc.

### Wet lab work commences and our real journey begins!

### Week 1 (20th to 26th May)

* Procurement of lab reagents from previous year's iGEM team and
  setting up the lab.
* LB media preparation and primary culture of *E.coli* DH5 - alpha.
* 24 hours growth curve analysis of *E.coli* secondary culture.

### Week 2 (27th May to 2nd June)

* Various genetic circuits to be used in the bacterial chassis were designed and ordered from Twist Bioscience.
* Different primers required for the PCR amplification of Biobricks and parts of genetic circuits were designed and ordered.

### Week 3 (3rd to 9th June)

* Glycerol stocks of *E.coli* DH5 - alpha were prepared.
* Competent cells were prepared using Calcium chloride method.
* Transformation was performed using the competent cell test kit, but the efficiency was very low.

### Week 4 (10th to 16th June)

* The 2nd batch of chemically competent cells was prepared and
  transformed with the competent cell test kit.
* This time, during the cell revival step of transformation, SOC
  medium was used instead of LB medium, but the efficiency was still very low.
* Resuspension of DNA samples from the 2019 iGEM DNA distribution
  kit: Constitutive promoter, an RBS site, Blue Fluorescent protein, Double terminator and an inducible RFP construct.
* Transformation was performed using the DNA parts and the competent cell kit was used as a control for the experiment.
* The transformation efficiency for the resuspended parts was less as compared to the competent cell kit.

**Collaboration**

* We had our first video conference with the iGEM team of MIT Pune, whom we are mentoring. We had an elaborate discussion regarding the details of each others’ projects and tried to solve several common wet lab problems that we had been facing.

### Week 5 (17th to 23rd June)

* The three solutions for the traditional method of plasmid isolation were prepared.
* Plasmids were isolated from the transformed colonies.
* Isolated colonies were run on 1% Agarose gel but there was a smear, suggesting the isolation had failed.
* Electrocompetent cells were prepared.

### Week 6 (24th to 30th June)

* Electrical transformation was done using a Bio-Rad electroporator to check the transformation efficiency. This was higher as compared to the chemical transformation.
* CCMB80 buffer preparation.
* The Hanahan method of preparing competent cells was used - the
  efficiency increased by 100 times as compared to the CaCl2
  competent cell.

### Week 7 (1st to 7th July)

* Colony screening of the transformed bacteria using colony PCR.
* DNA parts from Twist Bioscience were received.
* 3A assembly for circuit - 1 performed which consists of 1A, 1B and plasmid backbone. Part 1A had a NorR coding region under a constitutive promoter while part 1B has a pNO promoter which can be expressed only when Nsr binds to it in presence of Nitric oxide.
* No colonies obtained on the specific antibiotic plates.

### Week 8 (8th to 14th July)

* Double restriction of plasmid backbone pSB1C3, 1A and 1B parts
  using EcoRI and PstI.

* Setting up the ligation reaction of 3:1 and 5:1 insert: vector
  ratio using backbone as vector and individual parts as inserts.

**Human practices & Social awareness**

* We went to a local high school, Kalyani Central Model School, to
  conduct a workshop on genetic engineering and give them an informed
  view on the pros and cons of synthetic biology. Held a
  demonstration and interactive session of bacterial culturing and
  plating.
* Started preparing the presentation and designing the poster for all
  India iGEM meet 2019 conducted by IISER Bhopal.

### Week 9 (15th to 21st July)

* 3A assembly for the circuit - 3 performed with the upstream part
  being the pTet promoter from the DNA distribution kit, downstream
  part being 3C which had an NSR binding site with a GFP coding
  sequence and the pSB1C3 as the plasmid backbone.
* The ligated product was transformed and plated on CHM plates but no
  colonies were seen after 16 hours.
* After 24 hours, a few small colonies appeared for which colony PCR
  was performed using VF2 and VR primers and few of which showed
  positive results.
* Plasmid isolation of the same was done but the results were
  negative. We concluded that the colonies were foreign contaminants
  as they had whitish texture different from the E.coli and had no
  plasmid in them.

**Collaboration**

* Attended the All India iGEM Meet on the 20th and 21st of July held
  in IISER Bhopal.

### Week 10 (22nd to 28th July)

* Resuspension of pTet in pSB1C3 plasmid from the DNA distribution kit and transformation of the same in chemically prepared competent
  cells.
* Successful plasmid isolation was performed using Sigma Aldrich
  GenElute.
* Amplification of pTet from the isolated plasmid was performed using Biobrick Forward and Reverse primers.
* The expected band size of 133 bp was observed on 2% Agarose gel
  which was further eluted using Invitrogen Gel Elution Kit.

### Week 11 (29th July to 4th August)

* A colony PCR was done using colonies which have been transformed
  with circuits 1A (in pSB1C3 plasmid) and 1B (in pSB1A3 plasmid) and
  using VF2 and VR primers. Some of the PCR reactions gave a positive
  band corresponding to the respective sizes of 1A and 1B when run on
  a gel. However, when we tried to isolate the plasmids from these
  colonies we got negative results suggesting that the colony PCR was
  giving a pseudo positive result. On a closer analysis of the
  colonies we found that they were foreign contaminants.
* All DNA parts required for the constructing circuit 2 were diluted
  and PCR amplified using BBF and BBR primers. Circuit 2 consist of 5
  parts:

1. 2A - NorR coding sequence under a constitutive promotor
2. 2B - Tet R Coding Sequence under pNO promotor
3. 2C - pTET promotor
4. 2D - GFP coding sequence and Terminator site
5. Plasmid Backbone - pSB1C3

* In-circuit 2 NorR is constitutively produced, in the presence of a
  particular concentration of Nitric Oxide(NO) NorR along with NO
  binds to pNO promotor to produces pTET and inhibits the production
  of GPF. From this, we can quantify at what concentration of Nitric
  oxide pTET is produced.
* This Genetic circuit consists of many fragments, linear ligation of
  adjacent parts are done.
* 2A is digested with Spe1 and 2B is digested with Xba1 and performed
  linear ligation with a 1:1 ratio using T4 Ligase for 2hrs.
* The ligated product was purified on the 1.5% agarose gel, the
  desired band was not present.

### Week 12 (5th to 11th August)

* The eluted pTet PCR product was restricted using SpeI and PstI and the corresponding band of 89 bp was isolated by gel elution.
* Linearized pSB1C3 was restricted and purified using EcoRI and PstI.
* Purified 3C PCR was restricted using XbaI and PstI.
* A 3A assembly ligation protocol was followed to ligate all of them with pTet being the upstream part, and 3C being downstream.
* The ligation mixture was incubated overnight at 16°C followed by transformation with chemically competent cells.
* No colonies were seen by the end of 16 hours after plating on the specific antibiotic plates.
* New stock of CCMB 80 competent cells was prepared.
* 3A assembly of pTet and 3C in the linearized plasmid was repeated, but this time with a new batch of competent cells, but still there were no positive colonies.

### Week 13 (12th to 18th August)

* To pinpoint if the problem is in the ligation or in the individual restrictions, we switched to the linear ligation of DNA fragments.
* As the pTet part is very small in size, instead of using BBF and BBR primers we used BBR and VF2 primer which gave us an amplicon size of 223 bp.
* The newly amplified pTet was digested with SpeI and the 3C part was digested with XbaI. The ligation was set up at 37 °C for 2 hours.
* The ligated product was run on the gel and the five distinct bands could be seen clearly on the gel viz. self-ligation of 3C part, self-ligation of pTet, cross ligation of 3C and pTet, unligated 3c and unligated pTet, confirming the fact that both the restriction endonuclease and ligase are working properly.
* The cross ligated 3C and pTet was eluted and PCR was done on it
  using BBR and VF2 but it was seen that only the pTet part was
  amplified instead of the entire ligated product.

### Week 14 (19th to 25th August)

* The previous ligation was repeated with the linear ligation of 3A and pTet. Here, 3A (an NSR coding region under constitutive
  promoter ) was upstream to the pTet.
* This time we kept control with just the ligation of 3A alone and pTet alone. On running the ligated mixture on 2% agarose gel all ive expected bands could be seen. But the PCR amplification again gave amplified pTet alone instead of ligated 3A and pTet.
* On a thorough discussion with one of the professors, we realized that even though ideally the Biobrick suffix and prefix are cut with exonucleases at the site of SX ligation, but it still had some binding sites for the primers(~6bp), thus on amplification, due to stoichiometry the smaller sized product dominates.
* He further suggested us to follow the standard traditional way of inserting a DNA fragment in a plasmid.

### Week 15 (26th August to 1st September)

* The plasmid containing pTet was isolated and sequentially digested
  using XbaI and EcoRI, but due to very low concentration after two
  digestions, it was discarded.
* The plasmid containing pTet was doubly digested using XbaI and
  EcoRI.
* The 3A part was digested using XbaI and EcoRI.
* The ligation reaction was set up for 3:1 and 5:1 insert: vector
  ration where the pTet in the plasmid was vector.
* On transformation, there were no colonies on the antibiotic
  specific plates, but the control plate with pUC19 had many colonies
  suggesting there was no problem with the transformation protocol.

**Cell Culture**

* Cell Culture work started which involves Murine Macrophage J774A.1 cells and
  *Leishmania major* Culturing and Nitrc Oxide detection-based assays.
* US origin FBS was heat-inactivated, filter sterilized and aliquoted
  into 50ml to prevent contamination and ready to use.
* M199 media was prepared and added all other additional components
  required for culturing the Leishmania major and filter Sterilized.
* Phosphor buffer saline (PBS) was prepared and filter sterilized to
  make a cell culture grade PBS which is used in macrophage
  culturing.
* DMEM media was prepared and all other additional components were
  added to the media along with 10% FBS and filter sterilized.
* 15% heat-inactivated FBS was added to filter-sterilized M199 using
  a syringe filter.
* The cryopreserved *Leishmania major* were recovered and passaged.
* Cryopreserved Raw 267.3 macrophages were recovered and plated in
  60 mm Plate and latter passaged.
* *L.major*, Macrophage were cultured and confirmed that they are
  growing normally in their respective media prepared by us without
  any contamination.

### Week 16 (2nd to 8th September)

* DMEM media was prepared without adding any antibiotics.
* Experiments planned to infect the Murine Macrophage J774A.1 cells with *E.coli*
  Strain DH5-alpha
* Murine Macrophage J774A.1 cells are cultured in the 60 mm culture dish and
  after reaching 90% confluency, cells are splitted into two 35mm
  dishes, three 60 mm dishes.
* Coverslips were placed in p35 dish, cells were cultured in no
  antibiotic DMEM media and placed in an incubator at 37c with 5%
  CO2.
* Due to some technical problems, the incubator stopped working and
  cells died due to stress conditions.

**Human practices & Social awareness**

* The Human practice's team interviewed Dr. Nahid Ali, a prominent
  scientist working on leishmaniasis for decades, to gain some
  meaningful insights about the disease incidences around the country
  and its socio-economic impact.

### Week 17 (9th to 15th September)

* Part BBa\_K381001 with GFP under Pyear was resuspended from the DNA
  distribution kit and chemically competent cells were transformed
  using the diluted part.
* Pyear promoter is repressed by NSR protein in normal conditions,
  but in the presence of nitric oxide (NO), this repression is
  removed due to the binding of NO with NSR.
* We used Sodium Nitroprusside (SNP) as a NO donor for characterizing
  the promoter activity of Pyear.
* The secondary culture of transformed E. coli was induced with
  different concentrations of SNP ranging from 10-3 M to 10-6 M and
  the GFP production was quantified 6 hours after the induction using
  a microplate reader.

**Cell Culture**

* M199 media with different Hemin concentrations were prepared.
* For the proof of model that iron is indeed a major element for the
  growth of *Leishmania* and in its absence the growth can be hampered
  drastically, we designed an experiment where iron was a limiting
  factor for the growth.
* The cultures were grown in a series of decreasing Hemin
  concentration in the media as Hemin is the primary source of iron
  in cell culture media.
* *L.major* number was counted using a hemocytometer at different time
  points.
* The growth was observed over a period of 72 hours and the results
  were documented.
* Under Human Practices of iGEM, we made a google form and forwarded
  it among the common masses around the country to know the extent of
  awareness about the disease.

### Week 18 (16th to 22nd September)

* Mid-Semester Examinations!

### Week 19 (23rd to 29th September)

* 3C part was inserted into the pTet plasmid using traditional
  cloning techniques.
* The ligated product was used to transform the cells provided by
  NEB.
* Due to some technical problems, the incubator didn’t work overnight
  and the experiment failed.

**Collaboration**

* We collaborated with the iGEM teams of IISER Pune and REC Chennai to
  spread awareness about Leishmaniasis in their institutes.

**Human practices**

* We talked to Mr. Sourav a Ph.D. student working on the iron intake
  and metabolism of *Leishmania* who gave us enormous input about how
  we can modify our model to include the constraints of in-vitro
  applications.

### Week 20 (30th September to 6th October)

* BBa\_K861173 was resuspended from the iGEM distribution kit and
  transformed into the chemical competent cells.
* The part was characterized by growing it in M9 minimal media
  supplemented with a range of glucose and its analogs.
* The mRFP fluorescence was measured using a microplate reader and
  the results were documented.

### Week 21 (7th to 13th October)

* New IDT parts with complete G blocks arrived and we tried cloning
  it in the pSB1C3 backbone.

- We got the plasmid containing the Human Copper Transporter (hCT)
  protein cytoplasmic domain that can chelate divalent ions from the
  media.
- The protein expression was qualitatively compared between LB media
  and DMEM.
- The interaction of hCT with the Fe ions in PB buffer was quantified
  using Isothermal Titration Calorimetry.

**Cell culture**

* NO quantification experiment : Macrophage was infected with
  *Leishmania major* and using a Nitric Oxide assay kit we measure the
  change in NO concentration of Macrophage for 24 hours.

### Week 22 (14th to 20th October)

* Tried Gibson assembly for ligating the IDT parts in linearized
  backbone from iGEM.
* Working on the presentation and poster for the Giant Jamboree.
* Giving final touches to the Wiki pages.


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

# Team:IISER Kolkata/Experiments

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* [Home](https://2019.igem.org/Team%3AIISER_Kolkata)
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  + [Attributions](https://2019.igem.org/Team%3AIISER_Kolkata/Attributions)
* Project

  + [Description](https://2019.igem.org/Team%3AIISER_Kolkata/Description)
  + [Design](https://2019.igem.org/Team%3AIISER_Kolkata/Design)
  + [Experiments](https://2019.igem.org/Team%3AIISER_Kolkata/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AIISER_Kolkata/Notebook)
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  + [Results](https://2019.igem.org/Team%3AIISER_Kolkata/Results)
  + [Model](https://2019.igem.org/Team%3AIISER_Kolkata/Model)
* Parts

  + [Overview](https://2019.igem.org/Team%3AIISER_Kolkata/Parts)
  + [Basic](https://2019.igem.org/Team%3AIISER_Kolkata/Basic_Part)
  + [Composite](https://2019.igem.org/Team%3AIISER_Kolkata/Composite_Part)
  + [Collection](https://2019.igem.org/Team%3AIISER_Kolkata/Part_Collection)
* [Safety](https://2019.igem.org/Team%3AIISER_Kolkata/Safety)
* Humanity

  + [Overview](https://2019.igem.org/Team%3AIISER_Kolkata/Practices_Overview)
  + [Integrated HP](https://2019.igem.org/Team%3AIISER_Kolkata/Human_Practices)
  + [Engagement](https://2019.igem.org/Team%3AIISER_Kolkata/Public_Engagement)
  + [Social](https://2019.igem.org/Team%3AIISER_Kolkata/Social_Studies)

# Experiments

A great man once said, *"The true method of knowledge is through experiment."* And in the quest to gain knowledge we did a lot of experiments.

Most of our work dealt with genetically engineering our bacterial chassis i.e. *E. coli*. Additionally, to support our model and to gain some meaningful insights into the system, we did a variety of experiment involving Macrophage and *Leishmania major*.

### Bacterial Protocols

[# 1

##### LB Media Preparation](https://static.igem.org/mediawiki/2019/b/b9/T--IISER_Kolkata--1_bac.pdf)
[# 2

##### SOB Media Preparation](https://static.igem.org/mediawiki/2019/5/50/T--IISER_Kolkata--2_bac.pdf)
[# 3

##### 50X TAE Buffer](https://static.igem.org/mediawiki/2019/8/8d/T--IISER_Kolkata--3_bac.pdf)
[# 4

##### CCMB Buffer Preparation](https://static.igem.org/mediawiki/2019/b/bd/T--IISER_Kolkata--4_bac.pdf)
[# 5

##### Calcium chloride Competent Cell Preparation](https://static.igem.org/mediawiki/2019/f/fd/T--IISER_Kolkata--5_bac.pdf)
[# 6

##### Hanahan Competent Cell Preparation](https://static.igem.org/mediawiki/2019/4/45/T--IISER_Kolkata--6_bac.pdf)
[# 7

##### Electrocompetent Cell Preparation](https://static.igem.org/mediawiki/2019/5/52/T--IISER_Kolkata--7_bac.pdf)
[# 8

##### Heat Shock Transformation of Chemically Competent Cells](https://static.igem.org/mediawiki/2019/f/f5/T--IISER_Kolkata--8_bac.pdf)
[# 9

##### Transformation by Electroporation](https://static.igem.org/mediawiki/2019/a/ac/T--IISER_Kolkata--9_bac.pdf)
[# 10

##### PCR Amplification: Q5® H-F MM](https://static.igem.org/mediawiki/2019/2/23/T--IISER_Kolkata--10_bac.pdf)
[# 11

##### PCR Amplification: Taq Pol](https://static.igem.org/mediawiki/2019/2/28/T--IISER_Kolkata--11_bac.pdf)
[# 12

##### Restriction Digestion](https://static.igem.org/mediawiki/2019/1/16/T--IISER_Kolkata--12_bac.pdf)
[# 13

##### Ligation](https://static.igem.org/mediawiki/2019/4/40/T--IISER_Kolkata--13_bac.pdf)
[# 14

##### SDS-PAGE Electrophoresis](https://static.igem.org/mediawiki/2019/a/aa/T--IISER_Kolkata--14_bac.pdf)

### *Leishmania major* culture protocols

[# 1

##### M199 Preparation](https://static.igem.org/mediawiki/2019/2/25/T--IISER_Kolkata--cell_1.pdf)
[# 2

##### M199 Media with different Hemin Concentrations](https://static.igem.org/mediawiki/2019/b/b0/T--IISER_Kolkata--cell_2.pdf)
[# 3

##### Culturing *Leishmania major*](https://static.igem.org/mediawiki/2019/f/ff/T--IISER_Kolkata--cell_3.pdf)
[# 4

##### *Leishmania*-Macrophage Infection](https://static.igem.org/mediawiki/2019/2/23/T--IISER_Kolkata--cell_4.pdf)

### Macrophage culture protocols

[# 1

##### DMEM Culture Media Preparation](https://static.igem.org/mediawiki/2019/a/a2/T--IISER_Kolkata--cell_5.pdf)
[# 2

##### Phosphate Buffer Saline Preparation](https://static.igem.org/mediawiki/2019/9/90/T--IISER_Kolkata--cell_6.pdf)
[# 3

##### Murine Macrophage J774A.1 Culturing](https://static.igem.org/mediawiki/2019/d/d9/T--IISER_Kolkata--cell_7.pdf)

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

# Team:IISER Kolkata/Results

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* [Home](https://2019.igem.org/Team%3AIISER_Kolkata)
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  + [Attributions](https://2019.igem.org/Team%3AIISER_Kolkata/Attributions)
* Project

  + [Description](https://2019.igem.org/Team%3AIISER_Kolkata/Description)
  + [Design](https://2019.igem.org/Team%3AIISER_Kolkata/Design)
  + [Experiments](https://2019.igem.org/Team%3AIISER_Kolkata/Experiments)
  + [Notebook](https://2019.igem.org/Team%3AIISER_Kolkata/Notebook)
  + [Contribution](https://2019.igem.org/Team%3AIISER_Kolkata/Contribution)
  + [Results](https://2019.igem.org/Team%3AIISER_Kolkata/Results)
  + [Model](https://2019.igem.org/Team%3AIISER_Kolkata/Model)
* Parts

  + [Overview](https://2019.igem.org/Team%3AIISER_Kolkata/Parts)
  + [Basic](https://2019.igem.org/Team%3AIISER_Kolkata/Basic_Part)
  + [Composite](https://2019.igem.org/Team%3AIISER_Kolkata/Composite_Part)
  + [Collection](https://2019.igem.org/Team%3AIISER_Kolkata/Part_Collection)
* [Safety](https://2019.igem.org/Team%3AIISER_Kolkata/Safety)
* Humanity

  + [Overview](https://2019.igem.org/Team%3AIISER_Kolkata/Practices_Overview)
  + [Integrated HP](https://2019.igem.org/Team%3AIISER_Kolkata/Human_Practices)
  + [Engagement](https://2019.igem.org/Team%3AIISER_Kolkata/Public_Engagement)
  + [Social](https://2019.igem.org/Team%3AIISER_Kolkata/Social_Studies)

# Results

### New Parts for the Registry

We were successful in getting clones for two new BioBrick parts. One of them was a Nitric Oxide dependent device while the other was coding region for the metal ion chelator.

**Nitric Oxide sensing circuit**

We designed a new biobrick part (BBa\_K3011007)that can be activated in a certain concentration (10^-4 M to 10^-5 M) of Nitric Oxide in the media. Detailed information about the part can be found [here](http://parts.igem.org/Part%3ABBa_K3011007)

![](https://static.igem.org/mediawiki/2019/d/d2/T--iiser_kolkata--colony_pcr_ga.jpeg)

colony PCR was done to confirm the presence of correct transformants.

For colony PCR we used the vector specific VF2 and VR that when simulated on SnapGene showed a amplicon of 1730 bp. This was confirmed by the PCR products run on the gel. For the colonies with positive amplicon we grew them in liquid media until it reached an OD of around 0.6 and then it was induced by Sodium Nitroprusside solution which releases NO in aquatic media.

![](https://static.igem.org/mediawiki/2019/e/ec/T--iiser_kolkata--SNP_red_colony_induc.jpeg)

We induced the bacterial culture with 10^-4 M Nitric Oxide concentration and the induced colonies were producing mRFP after 6 hours.

We got our colony on the very last day so we couldn't manage to get a fluorescence data for the exact quantification of mRFP production but qualitatively it can be said that there is clear induction of Pyear promoter in the presence of Nitric Oxide in the medium. The concentration was determined from the characterization of part BBa\_K381001 with Pyear promoter controlling GFP production. The details of the same can be found on the [characterization page](https://2019.igem.org/Team%3AIISER_Kolkata/Contribution)

.

From the above observations we can clearly conclude that our designed BioBrick is correctly inserted in the PsB1c3 plasmid backbone and is expressed as expected.

**Metal Ion Chelator**

We used the cytoplasmic domain of Human Copper Transporter (hCtr1) as an analog of the metal ion chelator. In the original hypothesis, we proposed to use Aerobactin as our iron chelator but we had a limitation that- five different genes need to be synthesized to produce a functional Aerobactin molecule. Thus for all the experimental purposes, we planned to use hCTR1 which transports copper with high affinity in a time-dependent and saturable manner and is metal-specific. hCtr1-mediated copper transport is an energy-independent process and is stimulated by extracellular acidic pH and high K(+) concentrations. hCtr1 exists as a homomultimer (six domains)at the plasma membrane in mammalian cells. Moreover, it has been shown that hCtr1 is capable of binding to a variety of different divalent ions in the media. Thus here we are trying to show that this copper transporter has a capability to chelate iron too. For the expression of the protein in our bacteria, we are using only two domains. The part that we are using for our experimental purposes consists of only two homeodomains that can also efficiently bind to ions.

*Proteomic quantification*

The protein was inserted and expressed in the PEG expression vector. The bacteria with our final circuit will have to grow in conditions of macrophage culture. Thus in order to check the vibility of the protein expression we did a comparsion study to know the difference in protein expression in DMEM (cell culture media) as compared to LB media(bacterial). We confirmed the expression of the protein in the bacterial system by using an SDS PAGE electrophoresis.

![](https://static.igem.org/mediawiki/2019/6/68/T--iiser_kolkata--sds_page.jpeg)

SDS PAGE electrophoresis comparison of hCt cytoplasmic domain expression by transformed bacteria in LB media and DMEM. The last lane was loaded with the purified protein to check as a positive control.

The transformed bacteria were grown LB media and DMEM under identical conditions and from the figure, it is clearly seen that not only the protein is expressed in DMEM it also has much higher inducibility by IPTG in the mammalian cell culture media. The secondary culture was induced by 1mM of IPTG at 4 hours and the protein was isolated 6 hours post-induction.

*Binding Affinity to Iron*

To check the binding affinity of the Human copper transporter(hCt) cytoplasmic domain to free iron cations we used Isothermal titration calorimetry (ICT). It measures the heat absorbed or generated when ligand interacts with the protein and is used to determine the thermodynamic parameters of interactions in solution. It is most often used to study the binding of small molecules (such as inorganic ions ) to larger macromolecules (proteins.) In our case, it helped us to determine whether hCt is able to chelate free iron ions present in the surrounding environment.

![](https://static.igem.org/mediawiki/2019/7/7a/T--iiser_kolkata--itc_bvsb.jpeg)

ITC data for the buffer buffer interaction. The buffer used in this case was Tris(pH 8.0)

![](https://static.igem.org/mediawiki/2019/4/41/T--iiser_kolkata--itc_proteinVsfe.jpeg)

ITC data for the protein-ligand interaction. The protein being the hCtr1 and the ligand is Iron

From the graph obtained for the buffer-buffer interaction we can see that there is almost negligible energy change indicating the fact that there is no interaction within the different components of the buffer. On the other hand, for the energy graph obtained from the protein-ligand interaction, there is a considerable amount of energy change. Moreover, the thermodynamic parameter data (N1 and N2) given by the experiment suggests for every protein macromolecule there are two binding sites. This is in clear correlation with the expectations as there we are expressing only two of the homeodomains which in principle should have two binding domains.

### Supporting Experiments for the Model

We did a variety of experiments to quantify the growth dynamics and metabolism changes in the  *Lesihmania* and the macrophage in diffrent conditions.

**Effect of Hemin (Iron) on the growth of *Leishmania***

Iron is a necessary component for the growth of *Leishmania*. Hemin is one of the important sources of iron (Fe+3) for the invitro culture of *Leishmania*. The aim of this experiment was to test the effect on the growth of *Leishmania* if we decrease the amount of hemin (from its optimal) in the *Leishmania* culture media.

1. We made the *Leishmania* culture media (M199) without adding Hemin.
2. We made Hemin stock solution. Hemin is not soluble in water so we dissolve it in 1.4M NaOH solution to make the stock solution.
       Solubility of Hemin in 1.4M NaOH is 25 mg/mL.
       Molecular Weight of Hemin is 651.94 g/mol.
       We dissolved 25 mg of Hemin in 2 mL of 1.4M NaOH solution to make the stock of Hemin. So, the final concentration of Hemin stock solution is 0.02M.
3. Then we aliquoted the previously made M199 media in ten different falcon tubes and added different volume of Hemin (100% or the optimal concentration, 75% of optimal concentration, 50% of optimal concentration, 25% of optimal concentration, 0% or no Hemin) from the stock solution (two falcon tubes for each Hemin concentration).
4. Then we cultured *Leishmania major* in these media of different Hemin concentrations.
5. We counted the number of cells (in Hemocytometer) in every 24 hours for 3 days.

We started with the same number of cells in each culture media.

*24 hours later...*

![](https://static.igem.org/mediawiki/2019/3/36/T--IISER_Kolkata--DAY1_2.png)

*42 hours later...*

![](https://static.igem.org/mediawiki/2019/a/aa/T--IISER_Kolkata--DAY2_2.png
)

*72 hours later...*

![](https://static.igem.org/mediawiki/2019/b/bd/T--IISER_Kolkata--DAY3_2.png)

So, we can conclude due to the absence of Hemin the growth of *Leishmania* is affecting. Probably this growth is due to the iron which is present in FBS (used to prepare *Leishmania* culture media).

![](https://static.igem.org/mediawiki/2019/d/d5/T--IISER_Kolkata--overall_2.png)

**Nitric Oxide quantification in Stimulated Macrophage**

During any pathogenic infection, Macrophage gets activated to produce Nitric Oxide as an immune response to attract other immune cells to fight against the pathogen. Nitric Oxide has a very short half-life and freely diffusible, Abcam designed a detection kit to quantify nitric oxide inside the live cell.

This kit has reporter dye which binds to nitric oxide produced inside the live cells and exhibits orange fluorescence. Murine Macrophage J774A.1 cells were cultured in 60 mm culture dish were taken and seeded 4\*10^4 cells per well in 3 wells of 96 well plate, 3 wells with only media, 3 well seeded with 4\*10^4 cells and infected with 8\*10^5 *L.major*. In each well, both the dye and reaction buffer were added. The whole setup was incubated for 1 hour in CO2 incubator at 37 °C.

After 1hr the plate was placed in the microplate Varioskan LUX multimode microplate reader at 37 °C and kinetic loop of fluorescence readings were collected for 24 hours at intervel of 30min, at ex/em = 488/590nm.

![](https://static.igem.org/mediawiki/2019/7/75/T--IISER_Kolkata--Macrophage_NO.png)

From the graph we can observe, in normal macrophage, there is no increase or decrease in intracellular nitric oxide concentration. In macrophages infected with *L.major* up to 6 hours there no increase in intracellular nitric oxide concentration, this is because of macrophage starts engulfing the *L.major* after 6 hours, there is a sudden increase of intracellular nitric oxide concentration which is due to the phagocytosis of *L.major* by macrophages. After 20 hours, we can observe that there is a decrease in intracellular nitric oxide concentration, this is because *L.major* started regulating the production of intracellular nitric oxide production by inhibiting the iNOS production.