Team:IISER Kolkata/Results

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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

The transformed bacteria was grown until it reached an OD of around 0.6 and then it was induced by Sodium Nitroprusside solution which releases NO in aquatic media.

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

The plasmid was isolated and run on the agarose gel for further confirmation of the successful transformation.

The isolated plasmid was digested with EcoRI and PstI and we got a band at around confirming the insertion of the correct part in the PSB1C3 backbone.

Metal Ion Chelator

We used the cytoplasmic domain of Human Copper Transporter (hCtr1) as an analogue of the metal ion chelator. hCtr1 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 at the plasma membrane in mammalian cells. The part that we are using for our experimental purposes consists of only two domains that can bind ions.

Proteomic quantification

The protein was inserted and expressed in the PEG expression vector. We confirmed the expression of the protein in the bacterial system by using an SDS PAGE electrophoresis.

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.

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.

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

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...

Leishmania count after 24 hours in different Hemin concentration

42 hours later...

Leishmania count after 48 hours in different Hemin concentration

72 hours later...

Leishmania count after 72 hours in different Hemin concentration

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).

Growth of Leishmania in different Hemin concentration for three days

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 ex/em = 488/590nm.

Difference in Nitric oxide production between normal macrophage and macrophage infected with Leishmania (L.major)

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.