Immunometabolic Circuits in Infection for Advancing Host Directed Therapies

Summary

Here, a protocol for designing, constructing, and delivering tetracycline-controlled based inducible synthetic circuit is presented. The effect of the synthetic circuits on parasite elimination and cytokine expression can be studied by channelizing the presented pipeline. This protocol is relevant for researchers studying synthetic circuit-based therapeutics in infectious diseases.

Abstract

Immuno-metabolism is a pivotal determinant in the progression of leishmaniasis. Synthetic biology-based approach has garnered significant attention as a step toward therapeutic intervention targeting host-associated factors that drive leishmaniasis. Synthetic biology entails the engineering of genetic components in an orthogonal and modular manner to precisely modulate biological systems, imparting novel functions to cells. In the presented study, elucidation of a systematic pipeline for the development of an inducible tetracycline-controlled (TetON)-based synthetic circuit was aimed at delivering succinate dehydrogenase as a therapeutic agent to facilitate the elimination of intracellular Leishmania parasites. The outlined protocol describes the designing of a synthetic circuit and its subsequent validation. The proposed strategy also concentrates on the incorporation of synthetic circuits in the plasmid backbone as a delivery vehicle. Additionally, delivery machinery employing polyplexes-based nano-particles for the delivery of synthetic circuits was used in murine macrophage cell lines without compromising the cellular morphology. Standardization of the method was conducted for selecting transfected cells and determining optimal induction concentration for synthetic circuit expression. Observations reveal a distinct reduction in intracellular parasite burden in transfected cells compared to infected cells. Pro-inflammatory cytokines were expressed post-infection in synthetic circuit transfected and induced cells as a mechanism to promote parasite elimination. This underscores the synthetic biology-based method as a potent approach in leishmaniasis by targeting host factors associated with disease progression.

Introduction

The advancement of holistic, integrative medicine is a significant link that has been identified between metabolic alterations in immune cells and their ability to modulate immune cell phenotype in infectious diseases. Immuno-metabolism offers fresh perspectives for elucidating the pathogenesis of prevalent infectious diseases in humans. Moreover, it provides promising techniques for the development of vaccines and drugs by identifying novel targets for intervention¹. Metabolic reprogramming of immune cells is identified across various infectious diseases, including leishmaniasis. Leishmania spp. is responsible for causing leishmaniasis, of which Leishmania major (L. major) causes cutaneous leishmaniasis (CL). L. major parasites elicit a significant impact on the metabolism of host cells, particularly in macrophages, for proliferation and survival as intracellular parasites². Macrophages can typically polarize into either classically activated (M1) or alternatively activated (M2) subsets where M1 cells possess high pro-inflammatory cytokine secretion and production of nitric oxide (NO) through the metabolism of nitrogen species. M2 cells exhibit elevated levels of anti-inflammatory cytokines and display high arginase activity through nitrogen metabolism³. Thus, both phenotypes not only differ through their immune response but also in their metabolic pathways.

Systems biology of leishmaniasis aims to identify molecular targets for designing novel therapeutics through drug designing and development. Reconstruction of signaling networks and metabolic pathways aids in understanding leishmaniasis as a holistic model and draws insight for the identification of the principal component driving the diseased state. Mathematical modeling is one of the most favored applied analytical approaches for understanding the complexities of leishmaniasis⁴. Immunometabolic networks and mathematical models enabled the identification of succinate dehydrogenase (SDHA) as a key component of the M1 phenotype, which is expressed to eliminate the parasite⁵. Synthetic biology has applications in diagnostics, vaccine development and therapeutics in leishmaniasis. Metabolism targeting immune response modulation may be achieved through synthetic biology tools. For modulation of inositol phosphoryl ceramide synthase which regulates sphingolipid metabolism and alters host immune signaling, a genetic circuit that possessed bistable behavior was used to synthetically tune the intrinsic robustness of L. major infection model^6,7. Hence, systems and synthetic biology can provide a model-based genetic engineering platform for the development of precision medicine in the Leishmania model system.

M1 phenotype is marked by the expression of immuno-metabolic signaling networks where the production of inflammatory cytokines and metabolic pathways synergistically enhances parasite elimination. M1 polarization signifies the production of pro-inflammatory cytokines such as Interleukin-12 (IL-12), interferon-gamma (IFN-γ), and Tumor necrosis factor alpha (TNF-α). Metabolic pathways that are highly enriched in the M1 phenotype include glycolysis, pentose phosphate pathway, and truncated tricarboxylic acid cycle (TCA cycle)⁵. The truncated TCA cycle directs parasite elimination machinery through increased succinate and citrate levels, which guide the production of NO³. Accumulation of succinate by SDHA leads to activation of transcription factor hypoxia-inducible factor 1-alpha (HIF-1α), which induces the production of IL-1β⁸. In the M2 phenotype, fatty acid oxidation, increase in glutamine utilization, and enhanced oxidative phosphorylation of glucose are observed along with the production of anti-inflammatory cytokines such as Interleukin-10 (IL-10), Transforming growth factor beta (TGF-β), Interleukin-4 (IL-4) and Interleukin-13 (IL-13)³. M2 exhibits elongated mitochondria as a distinctive feature, leading to enhanced efficiency in energy generation. They utilize fatty acids, glucose, and glutamine as substrates to fuel the TCA cycle, thereby generating ATP through oxidative phosphorylation at a higher rate to promote parasite growth and proliferation⁹. SDHA enzyme responsible for succinate accumulation through truncated TCA can not only promote NO production but also initiate a succinate-dependent oxygen consumption through electron transport¹⁰. In Leishmania-infected macrophages, SDHA gets downregulated by 2.17 fold, indicating aerobic respiration might also be downregulated¹¹. In order to increase succinate consumption to induce pro-inflammatory cytokine-mediated NO production and promote parasite elimination, expression of SDHA is essential.

The synthetic circuit offers precision-targeted therapies that may alter the diseased system to achieve a bistable oscillation that may restore healthy phenotype at the spatiotemporal level. The synthetic circuit delivery and expression make the system dynamic and robust. One pivotal factor contributing to the advancement of synthetic biology lies in the ability to assemble genetic parts and elements in a modular fashion to enable the generation of intricate architectures and tailored functionalities¹². Constraints associated with traditional expression techniques have spurred the emergence of tetracycline operon-based inducible plasmid synthetic circuits, thus allowing rapid evaluation of plasmid and easy delivery across various cell types¹³. The tetracycline-responsive repressor (TetR) protein, when existing as a dimer, exhibits strong binding affinity to the tet operon (TetO), effectively suppressing transcription. However, upon the interaction of its ligand, doxycycline (dox), TetR undergoes a structural alteration, resulting in its dissociation from TetO, consequently allowing transcription to proceed unimpeded. This circuit can exhibit protein translation control¹⁴. Therefore, through the defined methodology, the designed tetracycline operon-based inducible plasmid synthetic circuit was enabled to express SDHA in the pEGFP-N1 vector. Evaluation of the transformation efficiency of the circuit and its yield provided insights into the competence of delivery. Through restriction digestion of the vector, the determination of the cloning efficiency was confirmed. Further, a transfected RAW264.7 mouse macrophage cell line derived from peritoneal macrophages of Balb/c mice was observed for doxycycline dose-dependent green fluorescent protein (GFP) expression in transfected cells. Additionally, the parasite elimination effect in transfected cells was corroborated with an expression of the synthetic circuit. Moreover, induction and expression of pro-inflammatory cytokines were observed, which was directed through an increase in the time point of infection in the synthetic circuit transfected and induced cells, which was directly associated with the functioning of the synthetic circuit.

Protocol

1. Cell culture of RAW264.7 cells

NOTE: Use low passage numbers for experimentation. RAW264.7 cell line was procured from National Cell repository of Biotechnology Research Innovation Council-National Centre for cell Sciences (BRIC-NCCS), Pune. As RAW264.7 cell line is a macrophage cell line it may start differentiating with increasing passages. After reviving the cells use the cell line for transfection after 2 passages.

1. Seed 5 x 10⁵ cells of the RAW264.7 cell line in a 25 cm² flask supplemented with 5 mL of Dulbecco's Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) and 100 I.U./mL penicillin as a complete medium.
2. Keep cells for growth and proliferation in an incubator with 5% CO₂ at 37 °C till 90% confluency is achieved.
3. Discard the media and wash 3x with 1x Phosphate buffer saline (PBS). Add 1 mL of PBS to the flask, scrape the cells using a scraper, and collect the cells in a 1.5 mL tube.
4. Centrifuge the cells at 300 x g for 5 min at 24 °C and resuspend the pellet using a 1 mL pipette in 1 mL of DMEM complete medium.
5. Count the cells in the cell counting chamber using 0.5% trypan blue to identify the percentage of live cells.

2. Cell culture of L. major parasite

NOTE: L. major promastigotes (MHOM/Su73/5ASKH) was a gift from BRIC-NCCS. Promastigotes should have a passage number less than 10. The lower passage number ensures the parasite is healthy and its ability to infect macrophages is higher.

1. Grow L. major promastigotes (MHOM/Su73/5ASKH) in Roswell Park Memorial Institute Medium (RPMI) supplemented with 20% FBS as a complete medium.
2. Seed 1 x 10⁶ promastigotes in non-vented 25 cm² flask containing 5 mL of complete medium. Incubate the flask at 27 °C for 5 days and observe the stationary phase promastigote L. major parasite at 20x in a bright field microscope.
   NOTE: Stationary phase promastigotes can be achieved in 4-7 days after culturing. They are characterized by their elongated flagellated state and reduced motility.

3. Designing a synthetic circuit in plasmid as a delivery vector

1. Designing parts of the synthetic circuit
   1. Open the Tinker cell software and proceed to the Parts tab. Select the Promoter component from the library and place it onto the canvas to symbolize the Cytomegalovirus (CMV) promoter within the synthetic circuit. Retrieve the repressor binding site, analogous to TetO, from the Parts tab and position it adjacent to the promoter on the canvas, integrating it seamlessly with the CMV promoter.
   2. Incorporate the tetracycline operator (TetO), Internal Ribosome Entry Sites (IRES), and the coding region by dragging and dropping repressor binding site, ribosome binding site (rbs) and coding region onto the canvas, integrating them with the existing genetic components.
   3. Rename the coding region as TetR, denoting the tetracycline-responsive repressor, which functions as a putative negative regulator within the synthetic circuit. Merge these genetic elements to facilitate the construction of the synthetic parts.
   4. Incorporate a spacer region (sd1) onto the canvas, designed to be cleavable by cellular proteases like porcine teschovirus-1 2A (P2A). Introduce an additional coding region representing the gene of interest, label it as SDHA, to the synthetic construct and integrate it with the other elements.
   5. Adjacent to the SDHA coding region, integrate another protease cleavable spacer region (sd2), which will be cleavable by cellular protease P2A. Introduce a coding region representing the reporter gene and label it as GFP into the construct.
      NOTE: GFP expression might provide an estimate for the production of SDHA and ensure that the synthetic circuit has a logical functioning¹³.
   6. Next to GFP, append a terminator (ter1) and verify the assembled parts by checking if all the parts are connected. The circuit must appear red upon clicking any part. This confirms the formation of a functional genetic circuit.
   7. From the Reaction tab, assign regulatory rates, which include parameters and initial values for protein production, to TetR, SDHA, and GFP to delineate the translation reaction within the synthetic circuit.
   8. Add a small molecule from the Parts tab, name it Doxycycline (dox), and assign a wave function to it by selecting Input from the Parts and Connection tab and then by clicking on Wave Input.
   9. From the Regulation tab, click on Repression Reaction. Drag and drop the Reaction Rate on canvas between dox and TetR. Set the reaction kinetics between dox and TetR by double clicking the Wave Function icon on dox and adjusting Doxyxycline.sin.amplitude to 10.
   10. Implement the transcriptional regulatory reaction between the TetR protein and the repressor binding site by selecting the reaction from the Regulation tab and placing it onto the canvas between TetR and TetO.
   11. By double-clicking each Component and Reaction, 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, which pops up with the graph, and observe the graph pattern for SDHA and TetR proteins.
2. Identification of genetic components from iGEM parts registry
   1. Open the Registry of Standard Biological parts (Table of Materials) and click on Search. Enter the name of the genetic component, e.g., CMV promoter.
      NOTE: The process for searching iGEM ID must be performed for all genetic components of the synthetic circuit. The steps for identifying the iGEM ID of the CMV promoter are enlisted; subsequently, the steps must be repeated for other components.
   2. To obtain the nucleotide sequence of the CMV promoter, click on the Get Part Sequence option and save the file as a text file.
      NOTE: It is crucial to note that stocks of all genetic parts are available.
   3. Repeat step 3.2.1 and step 3.2.2 for all genetic parts (tetracycline operator, Kozak sequence, TetR, P2A and GFP) except SDHA.
   4. To obtain protein coding sequence of SDHA, open NCBI and choose Nucleotide from the drop down next to search window. Type Succinate dehydrogenase and Mus musculus and search for nucleotide sequence.
   5. Click on the CDS option to get the coding sequence of SDHA and save the sequence in a text file.
   6. Merge the nucleotide sequence of all genetic regulatory parts in one file sequentially. Add start codon (ATG) right after the Kozak sequence and remove internal stop codons to avoid the formation of nascent polypeptides.
   7. Open ExPASy website¹⁵ and paste the nucleotide sequence. Click on Translate the Sequence option. Select 5'3' Frame 1 and remove the stop codons, which will be highlighted in red in the results section.
      NOTE: To identify if the nucleotide sequence is effectively translating into the desired polypeptide sequence, a translation tool such as ExPASy can be employed. This ensures the prevention of undesired nascent polypeptides. The selection of the pEGFP-N1 vector for expressing the synthetic circuit is optimal, given that the CMV promoter and GFP gene were pre-existing within the vector. All the genetic parts were assembled using a cloning protocol (which was outsourced). The restriction enzymes utilized for cloning the inserts are NotI and XhoI. These restriction enzyme sites are located within the multiple cloning site of the pEGFP-N1 vector.

4. Transformation of the synthetic circuit in Escherichia coli (E.coli) DH5α cells

1. Preparation of E.coli DH5α competent cells
   NOTE: Autoclaving the reagents is recommended. Reagents can be stored at 4 °C till use.
   1. Culture 1 mL of fresh inoculum in 50 mL of Luria Broth (LB) at 37 °C, 180 rpm. Grow the cells for approximately 2 h till an optical density (OD) of 0.4-0.6 is achieved at 600 nm.
   2. Centrifuge the cells at 4,696 x g at 4 °C for 15 min. Discard the supernatant manually in the discard flask by inverting the 50 mL centrifuge tube and harvesting the cell pellet.
   3. Gently resuspend the pellet in 2 mL of ice-cold 0.1 M Magnesium chloride (MgCl2) using a 1 mL pipette. Make up the volume up to 20 mL with ice-cold MgCl_2.
   4. Centrifuge the tube at 4,696 x g for 10 min at 4 °C and discard the supernatant. Gently resuspend the cells using a 1 mL pipette in 1mL of ice-cold 0.1M Calcium chloride (CaCl₂) and incubate on ice for 2 h. Use the freshly made competent cells for transformation.
2. Transformation of competent cells with synthetic circuit construct
   1. Measure 50 µL of competent cells and add the cells in a 1.5 mL tube. Place the tube on ice. Add 10 ng of synthetic circuit construct to the aliquot and keep on ice for 3 min.
   2. Heat shock cells at 42 °C for 3 min on a dry heating block. Once the incubation on the dry heating block is complete, place the cells back on ice for 3 min, add 1 mL of LB to the cells, and incubate at 37 °C for 1 h at 250 rpm.
   3. When the incubation is complete, centrifuge the cells at 4,696 x g at room temperature (RT) for 5 min and discard the supernatant using a 1 mL pipette. Resuspend the cells in 100 µL of LB and plate it on LB agar containing 50 µg/ mL kanamycin. Invert the plate and incubate at 37 °C for 24 h.

5. Isolation of synthetic circuit construct

1. In a 50 mL centrifuge tube, take 10 mL of LB medium and add 50 µg/ mL of kanamycin as a selection broth to culture transformed cells for plasmid isolation. Pick up a transformed colony using a 200 µL tip and add it to the selection broth.
2. Using an inoculating loop, select another single colony, streak a plate using the pentagonal technique, and incubate at 37 °C for 24 h in order to maintain single colony isolates.
3. Grow the cells overnight at 180 rpm at 37 °C. Next day, centrifuge the culture at 4,696 x g for 20 min at 24 °C. After centrifugation, discard the supernatant by inverting the tube and add 1 mL of resuspension buffer (50 mM Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), 10 mM Ethylenediamine tetraacetic acid (EDTA), 100 µg/ mL RNase A (pH-8.0)) to the pellet and resuspend the cells by gently pipetting.
   NOTE: To resuspension buffer add RNase A 5 min before adding the mix to the cell pellet.
4. Incubate the mix for 5 min at RT. Add 1 mL of lysis buffer (0.2 N Sodium Hydroxide (NaOH), 1 % Sodium Dodecyl Sulfate (SDS)) to the cells and mix by inverting the tube for 2 min. Incubate the tube for 5 min at RT.
5. Add 1 mL of neutralization buffer to the cells (3 M Potassium Acetate (CH₃CO₂K)) and mix by inverting the tube multiple times. Centrifuge the cells at 4,696 x g for 20 min at RT and collect the supernatant in a 1.5 mL tube.
6. Add 500 µL of isopropanol to supernatant and mix well by inverting. Incubate the supernatant on ice for 15 min and then centrifuge at 12,000 x g for 30 min at 4 °C.
   NOTE: Incubation can range from 5 min to 1h. During this, it is advised to gently mix the contents by inverting every 5 min.
7. Discard the supernatant, add 1 mL of 70% ethanol (chilled) to the pellet, and centrifuge at 12000 x g for 5 min at 4 °C. Repeat this step once again.
8. Airdry the pellet and resuspend the obtained plasmid in 20 µL of sterile nuclease-free water (NFW). Measure the concentration of plasmid on a spectrophotometer at 260/280 nm wavelength.
   NOTE: Add more NFW if the plasmid concentration is very high to be estimated through nanodrop. The process of plasmid dilution must be performed in a laminar hood.

6. Agarose gel electrophoresis (AGE) of synthetic circuit

1. Preparation of plasmid sample for AGE
   1. In a 1.5 mL tube, measure 20 µL of 1x Tris-acetate-EDTA (TAE). Add 4 µL of 6x loading buffer and mix well by pipetting.
   2. Give a quick spin to the tube, add 1 µg of the synthetic circuit plasmid vector and mix well by pipetting the mixture. This reaction mixture can be loaded on agarose gel.
      NOTE: Avoid rigorous pipetting as it may damage the plasmid structure. Instead give a quick spin for mixing reagents.
2. Restriction digestion of synthetic circuit
   1. In a 1.5 mL tube, add 2 µL of 10x restriction buffer, 1 µL of NotI, and 1 µL of XhoI restriction enzyme, and mix well by giving the contents a quick spin. Add 1 µg of synthetic circuit plasmid vector and give the contents a quick spin.
   2. Make up the volume of the tube to 20 µL by adding NFW to the contents and giving the tube a quick spin. Incubate the tube at 37 °C for 1 h.
   3. Post incubation, keep the tube at 70 °C for 15 min in a dry heating block to deactivate restriction enzymes. Cool the contents of the tube and add 6 µL of loading buffer to the reaction mixture. Mix the contents by giving the tube a quick spin. This reaction mixture can be loaded on agarose gel.
   4. Set up the agarose gel and run the gel using a previously described protocol¹⁶. Briefly, load 20 µL samples to each well, load 5 µL of 1 Kb DNA ladder, and run the gel at 100V. Disconnect the power supply after the electrophoresis is completed, acquire the gel on a gel imager, and turn on the fluorescence option to view the bands.

7. Transfection of synthetic circuit construct in RAW264.7 cell line

1. Preparation of samples
   1. Prepare samples to determine the parasite-eliminating effect by induced synthetic circuit. The first sample is untransfected RAW264.7 cells (control), the second is RAW264.7 cells infected with L. major promastigotes for 6 h (6 h Infected), third sample was RAW264.7 cells transfected with synthetic circuit and induced with dox (IMT), the infection time point samples are RAW264.7 cells transfected with synthetic circuit and induced with dox which is infected with L. major promastigotes (IMTI) for different time points 6 h (6 h IMTI), 12 h (12 h IMTI), 18 h (18 h IMTI) and 24 h (24 h IMTI).
   2. To prepare all samples, scrape the RAW264.7 cells from 25 cm² flask and centrifuge at 300 x g for 5 min at RT.
   3. Resuspend the pellet in 1 mL of DMEM complete medium. Take 10 µL of the resuspended cells in a 100 µL tube, add 10 µL of 0.5% trypan blue to the cells and mix well.
   4. Load 10 µL of cell and trypan blue mix on the cell counting chamber slide and take cell count from 4 chambers.
   5. Plate cells in coverslip bottom 96 well plates at 2 x 10⁴ cells per well and incubate the cells for 24 h. This sample is the control sample. This step remains constant for the preparation of other samples.
      NOTE: Pre-warm the media at 37 °C in a water bath before using.
   6. To prepare a 6 h Infected sample, centrifuge 1 mL of stationary phase L. major promastigotes at 7,273 x g for 10 min. Resuspend the pellet in 500 µL of DMEM complete medium and take the cell count on a cell counting chamber by following steps 7.1.3-7.1.4.
   7. Add 2 x 10⁵ L. major promastigotes to the well and incubate them with 5% CO₂ at 37 °C for 6 h. Post incubation, wash the cells 3x with PBS.
   8. To prepare the IMT sample, seed the cells by following steps (7.1.2-7.1.5) and perform transfection steps 7.2.1-7.2.6.
   9. To prepare samples of time points of IMTI, follow steps 7.1.2-7.1.5 and perform the transfection steps 7.2.1-7.2.6. Discard the DMEM complete media containing 500 µg/mL Geneticin (G418) and 1 µg/µL dox and wash cells 3x with 1x PBS.
   10. Add 2 x 10⁵ L. major promastigotes to the wells and incubate them for 6 h, 12 h, 18 h and 24 h. Post-infection, discard the promastigotes containing DMEM complete media and wash wells 3x with 1x PBS. Add 200 µL of DMEM complete media containing 500 µg/mL Geneticin (G418) and 1 µg/µL dox to the cells and incubate for 24 h.
2. Transfection of synthetic circuit
   1. In a 1.5 mL tube, add 24 µL of Reduced-Serum medium, which is an improved Minimal Essential Medium. To the media, add 1 µg of pEGFP-N1 vector cloned with synthetic circuit plasmid.
   2. In a fresh 1.5 mL tube, add 24 µL of Minimal Essential Medium and add 1 µL of Polyethylenimine (PEI; 1 µg /µL). Mix well by pipetting the mix at least 10x and incubate at RT for 5 min.
   3. Transfer the PEI mix to the 1.5 mL tube containing the DNA mix. Mix well by pipetting drop wise at least 10x. Incubate the DNA: PEI mix at RT for 10 min.
   4. Discard the media from the wells on the 96-well coverslip bottom plates and wash the cells 3x with PBS. Using a pipette, gently add the DNA:PEI dropwise to the cells and gently shake the plate. Incubate the cells with 5% CO₂ at 37 °C for 3 h.
   5. After incubation, add 200 µL of fresh Minimal Essential Medium to the cells, gently shake the plate, and incubate for 24 h with 5% CO₂ at 37 °C.
      NOTE: PEI is cytotoxic; hence, it is recommended to not incubate cells post transfection for more than 24 h.
   6. Next day, discard the media and wash cells 2x with PBS. Add DMEM complete media containing 500 µg/mL Geneticin (G418) and 1 µg/µL dox to the cells and incubate for 24 h.
3. Fixing the samples for confocal microscopy
   1. To fix all the samples, wash the cells with 1x PBS 3x, add 100 µL of 4% paraformaldehyde to the cells, and incubate at RT for 10 min.
   2. Wash the cells with PBS 3x. After washing is complete, add 1 µg/mL 4',6-diamidino-2-phenylindole (DAPI) to the cells and incubate at RT for 15 min in the dark.
   3. After incubation, wash the cells 3x with PBS. Observe the cells under a confocal microscope for GFP expression and DAPI staining.

8. Acquisition of transfected cells by confocal microscopy and quantification

1. Clean the coverslip bottom of the wells using absorbent tissue. Turn on the controls of the confocal laser scanning microscope. Next, turn on the compressor and mercury lamp.
2. To operate the machine, microscope controls, scan head control, laser key, laser module, lamp, microscope controller, and CPU must be turned on sequentially.
3. On the computer open the software (Table of Materials) and click on Enable XY Stage Control. A pop-up will open which will ask for executing cleaning for 7S3. Click on No. Close the live graph and adjust the setting screens.
4. To the 60x objective, add a drop of immersion oil, place the 96-well plate coverslip bottom plate on the stage, and move the stage in the X-Y plane to adjust the objective position on the well. Click on the Ocular and select Alexa-488 Channel. Click on Shutter Off to focus the cells on the eyepiece. Adjust the eyepiece by core and fine focusing.
5. Click on Shutter On present on the microscope control box and move the objective to adjust its position and focus the area of interest to observe the cells in Differential Interference Contrast (DIC). Switch to EPI mode on the microscope control box to observe the cells in the Alexa-488 wavelength filter for GFP expression. The cells should appear green.
6. Click on Shutter Off, go to LSM imaging, and choose the resolution for the image as 1,024 x 1,024. Click on Add Phase and drag to add a green channel as a phase.
7. On the Olympus program click on LSM and further click on Live Button to observe the cells in phase contrast and Alexa 488nm channel. Press Control and scroll to focus the cells from software for capturing images.
8. Adjust the gain and offset to adjust the fluorescence intensity captured by the detector. Click on Capture, name the images, and click on Save.
9. In imaging software, click on File, select all the images with the extension .oir, and click on Open.
10. Click on Tile View to visualize images in all channels. To add a scale bar to images, click on View and then choose the Scale bar option.
11. To save images from single channels as 16-bit grayscale, click on File and select Export Image. Select the image and check the Tick Marks on all channels. In the select frame tab in the split view option, choose Yes. In the output tab, choose the 24-bit RGB color with the burn-in info option and choose tiff as the file type for the image. In the export to folder, choose the appropriate file location to save the images and then click on OK.
12. To save merged images, follow the same steps, except, in the split view option, choose No, and in the output tab, choose Merged Channels with Burn Info.
13. To analyze the expression of GFP in cells, open Fiji in Windows and export the tiff file. Select the Color green to analyze GFP expression.
14. Select the cell area using the freehand tool option and click on Analyse to measure fluorescence intensity. Repeat the process for 3 images.
15. Export the data to an analysis software and perform statistical tests on the quantified data.

9. Parasite load assay

1. While visualizing the cells on a confocal microscope, count the total number of parasites present in individual macrophages.
2. Randomly examine 100 macrophages and note the number of intracellular parasites. Perfom statistical test between infected and time points of IMTI samples to examine the effect of SDHA expression on parasite count.

10. Quantification of IL-10 and IL-12 through Enzyme-Linked Immuno Sorbent Assay (ELISA)

1. Prepare samples as per steps 7.1 and 7.2 and collect the media in a 1.5 mL tube. Keep the media on ice or store it at -20 °C until further use. Follow the user kit manual instructions and perform ELISA for IL-10 and IL-12.

11. Cytokine profiling using Quantitative real-time reverse-transcription PCR (qRT-PCR)

1. Prepare the samples as per steps 7.1 and 7.2. Discard the supernatant using pipette and add 500 µL of TRIzol reagent and incubate the cells for 2 min at RT.
   NOTE: RNA isolation steps are to be performed in a laminar flow cabinet as a safety precaution against RNase contamination.
2. Collect the samples in a 1.5 mL tube, add 100 µL of chloroform to them, and mix by inverting the tubes 5x-8x. Incubate the samples for 15 min at RT. Centrifuge the samples at 12,000 x g for 15 min at 4 °C.
3. Three layers will be observed, collect the clear aqueous top layer in a fresh 1.5 mL tube. Add 500 µL of isopropanol to the aqueous layer, mix well by inverting 10x, and incubate the samples for 15 min at RT. After every 5 min mix the samples again by inverting the tubes.
4. Centrifuge the samples at 12,000 x g for 15 min at 4 °C. Discard the supernatant and wash the pellet 2x with 75% ethanol made in 0.1% Diethyl pyrocarbonate (DEPC) containing water for 5 min and centrifuge at 8,000 x g at 4 °C.
   NOTE: DEPC containing water degrades RNases, and therefore, it is recommended to use it for RNA isolation.
5. Lastly, air dry the pellet for 5 min at RT, dissolve it in 20 µL of DEPC containing water, and measure the concentration of isolated RNA, 260/280 and 260/230 ratio, using a spectrophotometer.
   NOTE: The 260/280 ratio should be approximately 2.0, which is generally indicative of a pure form of RNA, and the 260/230 ratio should be between 2.0-2.2 in order to consider that isolated RNA is free of unwanted chemical compounds such as TRIzole.
6. For preparation of 20 µL of each cDNA sample, thaw the reagents on ice and give them a short spin for 15 s at RT. Give a short spin to all the reagents and even to the RNA samples before use.
7. In an autoclaved 100 µL tube, take 0.8 µL of 25x dNTP, 2 µL of 10x Primer, 2 µL of 10x buffer. Add 1 µg/µL concentration of RNA and add 0.5 µL of reverse transcriptase enzyme. Make up the volume to 20 µL using DEPC containing water.
8. Give the cDNA samples a short spin for 1 min at RT. Keep the samples on ice.
9. Turn on the thermal cycler, unscrew the top panel to loosen the holder, and place the samples on the block. After placing the samples, screw the lid tight.
10. Once the system is on, click on Run. Select the Main option, then enter the Programs menu and select the CDNA option. Define the sample volume as 20 µL and click on RUN. The thermal cycler runs the program in four steps. The first step is 25 °C for 10 min, the second is 37 °C for 2 h, the third is 85 °C for 5 min, and the final step is 4 °C forever.
11. Once the cycle enters the fourth step, click on Enter and finish the cycle. Collect the samples and proceed for qRT-PCR. Store RNA samples at -80 °C and cDNA samples at -20 °C.
12. For detection of cytokines, thaw the required reagents 2x PCR Master mix, cDNA, and probes for TNF-α, IFN-γ, TGF-β, and NFW on ice.
    NOTE: All the reagents, reaction mixtures, and samples must be kept on ice.
13. In a 100 µL tube, prepare a cDNA sample by adding 1 µL of cDNA and 3.5 µL of NFW. To prepare the master mix, take 5 µL of master mix and add 0.5 µL of probe in a 100 µL tube. Mix the contents by giving a short spin. Calculate the number of samples and target genes and prepare the reaction mix accordingly.
    NOTE: Add NFW and master mix to the bottom of the 100 µL tube. Add cDNA and probe to the wall of the centrifuge tube as a precaution to ensure the volume is added and to avoid cross-mixing of any reagent.
14. Place the 8-well strips in the PCR cooler. Transfer 5.5 µL of master mix and probe-containing vial to the bottom of each well in the 8-well strips. Accordingly, repeat the step for all the target genes.
15. Transfer 4.5 µL of the cDNA and NFW mix to the wall of the well in 8-well strips. Accordingly, repeat the step for all the samples.
16. Seal the strips with the lids and give a short spin of 10 s to mix the genes and samples in the well. Place the strip in the qRT-PCR sample slot and close the slot.
17. On the desktop launch the software and login as a guest. A pop-up will appear on the screen. Click on Continue without Connection.
18. In the Setup menu, click on Advanced Setup. In Experiment Properties, write the name of the experiment in the Experiment Name tab. In what type of experiment do you want to set up option, select Quantitation-Comparative CT (ΔΔ CT).
19. In Which ramp speed do you want to use in the instrument? tab, click on Fast (~ 40 minutes to complete a run). In the plate setup menu, define gene names in the Define Targets option and type the sample names in Define Samples.
20. In Run Method tab, type 10 µL in Reaction Volume Per Well. Right before holding stage click on Add stage and select Holding stage. Change the temperature to 50 °C and time to 2 min. In the second holding stage do not make any changes, which will be 95 °C for 1 s.
21. In the cycling stage Step 1, change the temperature to 95 °C and time to 3 s. In step 2, change the temperature to 60 °C and time to 30 s. Click on Run to start the qRT-PCR cycle.

12. Statistical analysis

NOTE: For all data, the mean is represented as a bar, and the standard deviation represents the error bar in a bar graph (p-value< 0.05 is considered significant).

1. Open the data analysis software and insert the obtained data from the confocal experiment.
2. For confocal microscopy analysis, apply ordinary One-way ANOVA with Tukey's correction test for multiple group comparison for GFP expression. Consider the data with p < 0.05 as statistically significant. Prepare a graphical representation of the data with the mean represented as a bar and the standard deviation representing the error bar.
3. For parasite load assay, create a new sheet and enter the parasite count data for 100 macrophages. Apply ordinary One-way ANOVA with Tukey's correction between all samples. Consider the data with p-value< 0.05 as statistically significant.
4. For ELISA, perform Two-way ANOVA with Tukey's multiple comparisons test. For qRT-PCR, perform One-way ANOVA with Tukey's correction test.

Results

The arrangement of genetic parts forming the synthetic circuit for expressing the synthetic circuit is shown in (Figure 1A). The corresponding parts were cloned in pEGFP-N1 vector in an orthogonal and modular fashion, which is represented in Figure 1B. The simulation of the synthetic circuit revealed oscillatory dynamics in both SDHA and the tetracycline repressor. This observation suggests a reciprocal expression pattern characterized by periodic wave functions...

Discussion

The obtained results underscore the efficiency of the pipeline, which has been channelized for the design and implementation of a synthetic circuit. The construction of this synthetic circuit was a step towards developing precision medicine for infectious diseases like leishmaniasis. Through the in silico assembly of genetic parts and deterministic simulation, the distinguished modulatory effect of the TetON system was monitored. The effect of the circuit, notably, might have led to the reciprocal bistable expre...

Materials

Name	Company	Catalog Number	Comments
10 μL tips	Tarson	521050
10X restriction buffer	BioLabs	R0146S	Restriction digestion
1mL tips	Tarson	521016
1mL tube	Eppendorf	30121023
200 μL tips	Tarson	521010
25cm2 flask	Corning	430639	For cell culture
4',6-diamidino-2-phenylindole	ThermoFischer Scientific	D1306	nuclear staining
50mL tube	Corning	352070
60mm plates	Falcon	353002
6X TrackIt Cyan/Orange Loading buffer	ThermoFischer Scientific	10488085	Loading samples for AGE
96 well coverslip bottom plate	ThermoFischer Scientific	160376	For confocal imaging and transfection
96 well micro test plate flat bottom wells	Tarson	941196
Agarose	Sigma	A9539	For AGE
Agarose gel electrophoresis assembly	GeNei	93	For AGE
Bacterial laminar flow	ESCO Lifesciences	2120765	For transformation
Calcium chloride	Merck	C4901	Buffer /Reagent preparation
Cell culture laminar air flow	ThermoFischer Scientific	1323TS	For cell culture
Cell scraper	Genetix	90020	For cell culture
Cell spreader	Fischer Scientific	12822775
Centrifuge for 1.5mL tubes	Eppendorf	EP5404000537
Centrifuge for 50mL falcon	ThermoFischer Scientific	75004210
Chlorofrom	Fischer Scientific	67-66-3	For RNA isolation
CO2 incubator	ThermoFischer Scientific	3110	For cell culture
Cuvette	Eppendorf	6138000018	Estimation of plasmid concentration
CYTIVA IQ 500 gel imager	Cytiva	29655893	For AGE
DEPC TREATED H2O 1000 ML	ThermoFischer Scientific	750023	For RNA isolation
Doxycycline	Merck	33429	For induction of transfected cells
Dry heating block	Labnet		For transformation and inactivation of restriction enzymes
Dulbecco's Modified Eagle Medium	National centre for cell science		For cell culture
Ethanol	Sigma-Aldrich	1.00983	Buffer /Reagent preparation
Ethidium bromide	Sigma	E7637	For AGE
Ethylenediamine tetraacetic acid	Fischer Scientific	12635	Buffer /Reagent preparation
Evos brightfield microscope	ThermoFischer Scientific	AMEX1000
ExPasy	https://web.expasy.org/translate/		for contruction of synthetic circuit
Fetal Bovine Serum	Gibco	16000-044	For cell culture
FV31S-SW software	Olypmus		Image acquisition
Geneticin	Gibco	1563411	for transfection
Gibson assembly method for cloning	BIOMATIK		Construction of synthetic circuit
Graphpad prism	Graphpad		Statistical analysis
High-Capacity cDNA Reverse Transcription Kit	ThermoFischer Scientific	4368814	for cDNA synthesis
Isopropanol	Fischer Scientific	13825	Buffer /Reagent preparation
Kanamycin	Sigma	60615	For transformation and colony selection
Luria Bertini broth	Himedia	M1245	For culturing E.coli
Magnesium chloride	Fischer Scientific	BP214	Buffer /Reagent preparation
Micro Filt Vertical Laminar Air Flow	Microfilt
MicroAmp Fast 8-Tube Strip, 0.1 mL	ThermoFischer Scientific	4358293	For qRT-PCR
MicroAmp Optical 8-Cap Strips	ThermoFischer Scientific	4323032	For qRT-PCR
Microwave oven	LG	MC-7649DW	For AGE
Mm00434228_m1 (IFN-γ)	ThermoFischer Scientific	4331182	Taqman probes
Mm00443258_m1 (TNF)	ThermoFischer Scientific	4331182	Taqman probes
Mm01321739_m1(TGFB)	ThermoFischer Scientific	4331182	Taqman probes
Mouse ELISA Kit IL-10	ThermoFischer Scientific	BMS614	For ELISA
Mouse ELISA Kit IL-12	ThermoFischer Scientific	BMS616	For ELISA
Multiskan Sky with Touch Screen + μDrop Plate	ThermoFischer Scientific	51119600DP	For ELISA
MX-M Microplate Mixers 96-well Cell Culture Plate Mixer Adjustable 0-1500 rpm Laboratory Shaker Agitator	DIAB
National centre for Biotechnology Information	https://www.ncbi.nlm.nih.gov		for contruction of synthetic circuit
Neubauer's chamber	Marienfeld superior	640010	For cell count
Non-vented 25cm2 flask	Corning	353014	For culturing parasite
NotI	BioLabs	R3189M	Restriction enzyme
Nuclease free water	Biodesign	1QIA	Reagent preparation
Olympus Cell Sens Dimension Desktop 2.3 software	Olypmus		Image processing
Olympus FV 3000	Olympus		For confocal imaging
OPTI-MEM media	Gibco	31985070	media for Transfection
pEGFP-N1 vector cloned with synthetic circuit	Biomatik
Penicillin	ThermoFischer Scientific	15070063	For cell culture
Polyethylenimine	MERCK	764965	for transfection
Potassium Acetate	Fischer Scientific	YBP364500	Buffer /Reagent preparation
Potassium Chloride	Fischer Scientific	BP366	Buffer /Reagent preparation
Potassium Phosphate Monobasic	Sigma	P5655	Buffer /Reagent preparation
Power supply pack	BIORAD	1645070	For AGE
Registry of Standard Biological parts	https://parts.igem.org/Main_Page		for contruction of synthetic circuit
RNAiso Plus	Takara	38220090	For RNA isolation
RNase A	ThermoFischer Scientific	12091021	Buffer /Reagent preparation
Roswell Park Memorial Institute Medium	National centre for cell science		For culturing parasite
Shaker incubator	REMI	CIS 24 plus	For culturing E.coli
Sodium chloride	Qualigens	Q27605	Buffer /Reagent preparation
Sodium Dodecyl Sulfate	Sigma-Aldrich	L3771	Buffer /Reagent preparation
Sodium Hydroxide	Fischer Scientific	15895	Buffer /Reagent preparation
Sodium Phosphate Dibasic	Sigma	S5136	Buffer /Reagent preparation
Spectrophotometer	Eppendorf		Estimation of plasmid concentration
Spin win	Trason	1020
StepOne plus Real Time PCR system	Life technologies	272006777	For qRT-PCR
StepOne software version 2.3	Life technologies		For qRT-PCR
TaqMan Fast Universal PCR Master Mix (2X), no AmpErase UNG	ThermoFischer Scientific	4366072
Tinker cell			Synthetic circuit construction and simulation
TrackIt 1 Kb Plus DNA ladder	ThermoFischer Scientific	10488085	For AGE
Tris (hydroxymethyl) aminomethane hydrochloride	Himedia	MB029	Buffer /Reagent preparation
Tris-acetate-EDTA	SERVA	42549.1	Buffer /Reagent preparation
Trypan blue	Sigma	T8154	For cell count
XhoI	BioLabs	R0146S	Restriction enzyme