Team:IISER Kolkata/Results
Results
New Parts for the registry
We were succesful 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^-4M to 10^-5M) 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.
Fig 1: We induced the bacterial culture with 10^(-4M) Nitric Oxide concentration and the induced colonies were producing mRFP after 4 hours.
Fig 2: The plasmid was isolated and run on the agarose gel for further confirmation of the successful transformation.
Fig 3: 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.
Fig 1: 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.
Fig 2:
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.
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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 Wight 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).
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Then we cultured Leishmania major in these media of different Hemin concentrations.
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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).
