Systems biology offers insight into the biological networks and mechanism in infection models, while synthetic biology enables precision therapy. We are exploring the immunometabolism in Leishmania major infection and the potential of biosynthetic circuits for disease resolution, revolutionizing immunotherapeutics. In recent years, synthetic biology have shown great promise towards the development of newer and effective strategies to combat Leishmaniasis.
Some of the recent developments are in the field of engineering synthetic cytokines, bistable synthetic circuit CRISPR-Cas9, and synthetic peptides are being investigated as therapeutics. Our protocol targets the host protein, which is modulated by the parasite for its survival. Thus we study the effect on the circuit not only on the parasite, but also on the host.
Moreover, we studied the effect of synthetic circuit on addressing orthogonality and modularity function upon delivery in the host. To begin, on a computer, open the TinkerCell software and click the parts tab. Select the CMV promoter component from the library and place it onto the canvas to symbolize the cytomegalovirus promoter within the synthetic circuit.
Then retrieve the repressor binding site analogous to the tetracycline operator and position it adjacent to the promoter on the canvas, integrating with the CMV promoter. Drag and drop the internal ribosome entry sites and the coding region onto the canvas, integrating them with the existing genetic components. Rename the coding region as tetracycline-responsive repressor.
Merge these genetic elements to facilitate the construction of the synthetic parts. Incorporate a spacer region cleavable by porcine teschovirus 12A onto the canvas. Introduce an additional coding region, succinate dehydrogenase or SDHA, representing the gene of interest in the synthetic construct and integrating it with the other elements.
Adjacent to the SDHA coding region, integrate another spacer region cleavable by cellular protease porcine teschovirus 12A. Introduce a coding region representing the reporter gene and label it as GFP into the construct. Next to GFP, append a terminator.
To verify the formation of a functional genetic circuit, click on any part of the circuit. The circuit appears red upon clicking. From the reaction tab, assign regulatory rates to tetracycline-responsive repressor, SDHA, and GFP to delineate the translation reaction within the synthetic circuit.
Add a small molecule from the parts tab and name it doxycycline. To assign a wave function to doxycycline, from the parts and connection tab, select input, and click on wave input. From the reaction tab, click on repression reaction.
Drag and drop the reaction rate on canvas between dox and tetracycline-responsive repressor. To set the reaction kinetics between dox and tetracycline responsive repressor, double-click the wave function icon on dox and adjust doxycycline.sin. amplitude to 10.
To implement the transcriptional regulatory reaction between the tetracycline-responsive repressor protein and the repressor binding site, select the reaction from the reaction tab and place it onto the canvas between the tetracycline-responsive repressor and tetracycline operator. Double-click each component and reaction to set an initial concentration and parameter for simulation. Click on simulation, choose deterministic, and simulate the circuit for 100 time units.
Adjust the simulation graph from the control panel and observe the graph pattern for SDHA and tetracycline-responsive repressive proteins. Open the registry of standard biological parts, then click on search and enter the name of the genetic component. To obtain the nucleotide sequence of the CMV promoter, click on the get part sequence and save the file as a text file.
To obtain the protein coding sequence of SDHA, open NCBI and choose nucleotide from the dropdown next to the search window. Type succinate dehydrogenase and Mus musculus and search for nucleotide sequence. Click on the CDS option to get the coding sequence of SDHA and save the sequence in a text file.
Sequentially merge the nucleotide sequence of all genetic regulatory parts in one file. Add a start codon ATG right after the Kozak sequence and remove internal stop codons to avoid the formation of nascent polypeptides. Open the Expasy website and paste the nucleotide sequence.
Click on translate the sequence option, then select five prime three prime frame one and remove the stop codons. To begin, culture RAW264.7 cells at 37 degrees Celsius until 90%confluency is achieved. Prepare the following samples to determine the parasite-eliminating effect of the induced synthetic circuit.
Scrape the RAW264.7 cells from a 25 square centimeter flask. Centrifuge the cell suspension at 300G for five minutes and re-suspend the pellet in one milliliter of DMEM complete medium. Transfer 10 microliters of the re-suspended cell suspension into a 100-microliter tube.
Add 10 microliters of 0.5%trypan blue to the cells and mix well. Load 10 microliters of cell and trypan blue mix on the cell counting chamber slide and take cell count from four chambers. Plate two times 10 to the power of four cells per well in cover slip bottom 96-well plate and incubate at 37 degrees Celsius with 5%carbon dioxide for 24 hours.
To prepare a six hours infected sample, centrifuge one milliliter of stationary phase Leishmania major promastigotes at 7, 273G for 10 minutes and re-suspend the pellet in 500 microliters of DMEM complete medium. After counting, add two times 10 to the power of five L.major promastigotes in cover slip bottom 96-well plate. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for six hours.
Post-incubation, wash the cells three times with PBS. To prepare the IMT and IMTI samples, grow two times 10 to the power of five L.major promastigotes for 6, 12, 18, and 24 hours. In a 1.5-milliliter tube, add 24 microliters of reduced serum medium and one microgram of synthetic circuit plasmid.
In another 1.5-milliliter tube, add 24 microliters of reduced serum medium and one microliter of polyethylenimine or PEI. I mix well by pipetting at least 10 times and incubate at room temperature for five minutes. Transfer the PEI mix to the 1.5-milliliter tube containing the DNA and mix well.
Incubate the tube for 10 minutes. Discard the media from the wells on the 96-well cover slip bottom plate and wash the cells three times with PBS. Using a pipette, dropwise add the DNA-PEI mix to the cells and gently shake the plate.
Incubate the cells at 37 degrees Celsius with 5%carbon dioxide for three hours. Next, add 200 microliters of fresh reduced serum medium to the cells, gently shake the plate, and incubate for 24 hours at 37 degrees Celsius with 5%carbon dioxide. The following day, discard the medium.
After washing the cells twice with PBS, add DMEM complete media containing geneticin and doxycycline to the cells and incubate for 24 hours. Simulation of the synthetic circuit revealed oscillatory dynamics in both SDHA and the tetracycline repressor. The introduction of the synthetic circuit into Escherichia coli DH5-alpha resulted in transformed colonies exhibiting resistance to kanamycin, indicative of efficient transformation.
Transfection with the synthetic circuit and induction with doxycycline significantly increased GFP expression in IMT cells over time. Post-transfection and doxycycline induction, a significant reduction in intracellular parasite load was observed in infected macrophages. Cytokine levels of interleukin 10 and interleukin 12 were highest at six hours post-infection, with no significant difference observed upon transfection and induction of synthetic circuits.
A significant increase in tumor necrosis factor alpha, interferon gamma, and transforming growth factor beta was observed in the 24-hour IMTI sample, indicating the upregulation of pro-inflammatory cytokines.
