Team:IISER Kolkata/Results
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
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.
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
.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.
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.
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.
- We made the Leishmania culture media (M199) without adding Hemin.
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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.
- 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).
- Then we cultured Leishmania major in these media of different Hemin concentrations.
- 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...
42 hours later...
72 hours later...
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).
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.
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.