The authors would like to thank the Director of the Biotechnology Research and Innovation Council- National Centre for Cell Science (BRIC-NCCS), Pune for supporting our research and the BRIC-NCCS Bioinformatics Facility.

Designing Tetracycline-Controlled Synthetic Circuits for Leishmaniasis Therapy

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+From+systems+biology+to+improve+knowledge+of+Leishmania+drug+resistance.">Perspectives From systems biology to improve knowledge of Leishmania drug resistance.</a> Front Cell Infection Microbiol. 11, 653670 (2021).</li><li>Khandibharad, S., Singh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immuno-metabolic+signaling+in+leishmaniasis:+insights+gained+from+mathematical+modeling.">Immuno-metabolic signaling in leishmaniasis: insights gained from mathematical modeling.</a> Bioinfo Adv. 3 (1), vbad125 (2023).</li><li>Mandlik, V., Limbachiya, D., Shinde, S., Mol, M., Singh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+circuit+of+inositol+phosphorylceramide+synthase+in+Leishmania+a+chemical+biology+approach.">Synthetic circuit of inositol phosphorylceramide synthase in Leishmania a chemical biology approach.</a> J Chem Biol. 6 (2), 51-62 (2013).</li><li>Khandibharad, S., Singh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+biology+for+combating+leishmaniasis.">Synthetic biology for combating leishmaniasis.</a> Front Microbiol. 15, 1338749 (2024).</li><li>Volpedo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+of+live+attenuated+centrin+gene-deleted+Leishmania+vaccine+candidates.">The history of live attenuated centrin gene-deleted Leishmania vaccine candidates.</a> Pathogens. 11 (4), 431 (2022).</li><li>Almeida, F. 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The authors have nothing to disclose.

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...

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 model6,7. 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-&#947;), and Tumor necrosis factor alpha (TNF-&#945;). 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-1&#945;), which induces the production of IL-1&#946;8. 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-&#946;), 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 &#956;L tips</td>
			<td>Tarson</td>
			<td>521050</td>
			<td></td>
		</tr>
		<tr>
			<td>10X restriction buffer</td>
			<td>BioLabs</td>
			<td>R0146S</td>
			<td>Restriction digestion</td>
		</tr>
		<tr>
			<td>1mL tips</td>
			<td>Tarson</td>
			<td>521016</td>
			<td></td>
		</tr>
		<tr>
			<td>1mL tube</td>
			<td>Eppendorf</td>
			<td>30121023</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L tips</td>
			<td>Tarson</td>
			<td>521010</td>
			<td></td>
		</tr>
		<tr>
			<td>25cm2 flask</td>
			<td>Corning</td>
			<td>430639</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>4&#39;,6-diamidino-2-phenylindole</td>
			<td>ThermoFischer Scientific</td>
			<td>D1306</td>
			<td>nuclear staining</td>
		</tr>
		<tr>
			<td>50mL tube</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>60mm plates</td>
			<td>Falcon</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>6X TrackIt Cyan/Orange Loading buffer</td>
			<td>ThermoFischer Scientific</td>
			<td>10488085</td>
			<td>Loading samples for AGE</td>
		</tr>
		<tr>
			<td>96 well coverslip bottom plate</td>
			<td>ThermoFischer Scientific</td>
			<td>160376</td>
			<td>For confocal imaging and transfection</td>
		</tr>
		<tr>
			<td>96 well micro test plate flat bottom wells</td>
			<td>Tarson</td>
			<td>941196</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>A9539</td>
			<td>For AGE</td>
		</tr>
		<tr>
			<td>Agarose gel electrophoresis assembly</td>
			<td>GeNei</td>
			<td>93</td>
			<td>For AGE</td>
		</tr>
		<tr>
			<td>Bacterial laminar flow</td>
			<td>ESCO Lifesciences</td>
			<td>2120765</td>
			<td>For transformation</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Merck</td>
			<td>C4901</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Cell culture laminar air flow</td>
			<td>ThermoFischer Scientific</td>
			<td>1323TS</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Genetix</td>
			<td>90020</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Cell spreader</td>
			<td>Fischer Scientific</td>
			<td>12822775</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge for 1.5mL tubes</td>
			<td>Eppendorf</td>
			<td>EP5404000537</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge for 50mL falcon</td>
			<td>ThermoFischer Scientific</td>
			<td>75004210</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorofrom</td>
			<td>Fischer Scientific</td>
			<td>67-66-3</td>
			<td>For RNA isolation</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>ThermoFischer Scientific</td>
			<td>3110</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Cuvette</td>
			<td>Eppendorf</td>
			<td>6138000018</td>
			<td>Estimation of plasmid concentration</td>
		</tr>
		<tr>
			<td>CYTIVA IQ 500 gel imager</td>
			<td>Cytiva</td>
			<td>29655893</td>
			<td>For AGE</td>
		</tr>
		<tr>
			<td>DEPC TREATED H2O 1000 ML</td>
			<td>ThermoFischer Scientific</td>
			<td>750023</td>
			<td>For RNA isolation</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Merck</td>
			<td>33429</td>
			<td>For induction of transfected cells</td>
		</tr>
		<tr>
			<td>Dry heating block</td>
			<td>Labnet</td>
			<td></td>
			<td>For transformation and inactivation of restriction enzymes</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>National centre for cell science</td>
			<td></td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>1.00983</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Sigma</td>
			<td>E7637</td>
			<td>For AGE</td>
		</tr>
		<tr>
			<td>Ethylenediamine tetraacetic acid</td>
			<td>Fischer Scientific</td>
			<td>12635</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Evos brightfield microscope</td>
			<td>ThermoFischer Scientific</td>
			<td>AMEX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>ExPasy</td>
			<td>https://web.expasy.org/translate/</td>
			<td></td>
			<td>for contruction of synthetic circuit</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>FV31S-SW software</td>
			<td>Olypmus</td>
			<td></td>
			<td>Image acquisition</td>
		</tr>
		<tr>
			<td>Geneticin</td>
			<td>Gibco</td>
			<td>1563411</td>
			<td>for transfection</td>
		</tr>
		<tr>
			<td>Gibson assembly method for cloning</td>
			<td>BIOMATIK</td>
			<td></td>
			<td>Construction of synthetic circuit</td>
		</tr>
		<tr>
			<td>Graphpad prism</td>
			<td>Graphpad</td>
			<td></td>
			<td>Statistical analysis</td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFischer Scientific</td>
			<td>4368814</td>
			<td>for cDNA synthesis</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fischer Scientific</td>
			<td>13825</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Sigma</td>
			<td>60615</td>
			<td>For transformation and colony selection</td>
		</tr>
		<tr>
			<td>Luria Bertini broth</td>
			<td>Himedia</td>
			<td>M1245</td>
			<td>For culturing E.coli</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fischer Scientific</td>
			<td>BP214</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Micro Filt Vertical Laminar Air Flow</td>
			<td>Microfilt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Fast 8-Tube Strip, 0.1 mL</td>
			<td>ThermoFischer Scientific</td>
			<td>4358293</td>
			<td>For qRT-PCR</td>
		</tr>
		<tr>
			<td>MicroAmp Optical 8-Cap Strips</td>
			<td>ThermoFischer Scientific</td>
			<td>4323032</td>
			<td>For qRT-PCR</td>
		</tr>
		<tr>
			<td>Microwave oven</td>
			<td>LG</td>
			<td>MC-7649DW</td>
			<td>For AGE</td>
		</tr>
		<tr>
			<td>Mm00434228_m1 (IFN-&#947;)</td>
			<td>ThermoFischer Scientific</td>
			<td>4331182</td>
			<td>Taqman probes</td>
		</tr>
		<tr>
			<td>Mm00443258_m1 (TNF)</td>
			<td>ThermoFischer Scientific</td>
			<td>4331182</td>
			<td>Taqman probes</td>
		</tr>
		<tr>
			<td>Mm01321739_m1(TGFB)</td>
			<td>ThermoFischer Scientific</td>
			<td>4331182</td>
			<td>Taqman probes</td>
		</tr>
		<tr>
			<td>Mouse ELISA Kit IL-10</td>
			<td>ThermoFischer Scientific</td>
			<td>BMS614</td>
			<td>For ELISA</td>
		</tr>
		<tr>
			<td>Mouse ELISA Kit IL-12</td>
			<td>ThermoFischer Scientific</td>
			<td>BMS616</td>
			<td>For ELISA</td>
		</tr>
		<tr>
			<td>Multiskan Sky with Touch Screen + &#956;Drop Plate</td>
			<td>ThermoFischer Scientific</td>
			<td>51119600DP</td>
			<td>For ELISA</td>
		</tr>
		<tr>
			<td>MX-M Microplate Mixers 96-well Cell Culture Plate Mixer Adjustable 0-1500 rpm Laboratory Shaker Agitator</td>
			<td>DIAB</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>National centre for Biotechnology Information</td>
			<td>https://www.ncbi.nlm.nih.gov</td>
			<td></td>
			<td>for contruction of synthetic circuit</td>
		</tr>
		<tr>
			<td>Neubauer&#39;s chamber</td>
			<td>Marienfeld superior</td>
			<td>640010</td>
			<td>For cell count</td>
		</tr>
		<tr>
			<td>Non-vented 25cm2 flask</td>
			<td>Corning</td>
			<td>353014</td>
			<td>For culturing parasite</td>
		</tr>
		<tr>
			<td>NotI</td>
			<td>BioLabs</td>
			<td>R3189M</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Nuclease free water</td>
			<td>Biodesign</td>
			<td>1QIA</td>
			<td>Reagent preparation</td>
		</tr>
		<tr>
			<td>Olympus Cell Sens Dimension Desktop 2.3 software</td>
			<td>Olypmus</td>
			<td></td>
			<td>Image processing</td>
		</tr>
		<tr>
			<td>Olympus FV 3000</td>
			<td>Olympus</td>
			<td></td>
			<td>For confocal imaging</td>
		</tr>
		<tr>
			<td>OPTI-MEM media</td>
			<td>Gibco</td>
			<td>31985070</td>
			<td>media for Transfection</td>
		</tr>
		<tr>
			<td>pEGFP-N1 vector cloned with synthetic circuit</td>
			<td>Biomatik</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>ThermoFischer Scientific</td>
			<td>15070063</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>MERCK</td>
			<td>764965</td>
			<td>for transfection</td>
		</tr>
		<tr>
			<td>Potassium Acetate</td>
			<td>Fischer Scientific</td>
			<td>YBP364500</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fischer Scientific</td>
			<td>BP366</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Potassium Phosphate Monobasic</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Power supply pack</td>
			<td>BIORAD</td>
			<td>1645070</td>
			<td>For AGE</td>
		</tr>
		<tr>
			<td>Registry of Standard Biological parts</td>
			<td>https://parts.igem.org/Main_Page</td>
			<td></td>
			<td>for contruction of synthetic circuit</td>
		</tr>
		<tr>
			<td>RNAiso Plus</td>
			<td>Takara</td>
			<td>38220090</td>
			<td>For RNA isolation</td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>ThermoFischer Scientific</td>
			<td>12091021</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute Medium</td>
			<td>National centre for cell science</td>
			<td></td>
			<td>For culturing parasite</td>
		</tr>
		<tr>
			<td>Shaker incubator</td>
			<td>REMI</td>
			<td>CIS 24 plus</td>
			<td>For culturing E.coli</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Qualigens</td>
			<td>Q27605</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fischer Scientific</td>
			<td>15895</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic</td>
			<td>Sigma</td>
			<td>S5136</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Estimation of plasmid concentration</td>
		</tr>
		<tr>
			<td>Spin win</td>
			<td>Trason</td>
			<td>1020</td>
			<td></td>
		</tr>
		<tr>
			<td>StepOne plus Real Time PCR system</td>
			<td>Life technologies</td>
			<td>272006777</td>
			<td>For qRT-PCR</td>
		</tr>
		<tr>
			<td>StepOne software version 2.3</td>
			<td>Life technologies</td>
			<td></td>
			<td>For qRT-PCR</td>
		</tr>
		<tr>
			<td>TaqMan Fast Universal PCR Master Mix (2X), no AmpErase UNG</td>
			<td>ThermoFischer Scientific</td>
			<td>4366072</td>
			<td></td>
		</tr>
		<tr>
			<td>Tinker cell</td>
			<td></td>
			<td></td>
			<td>Synthetic circuit construction and simulation</td>
		</tr>
		<tr>
			<td>TrackIt 1 Kb Plus DNA ladder</td>
			<td>ThermoFischer Scientific</td>
			<td>10488085</td>
			<td>For AGE</td>
		</tr>
		<tr>
			<td>Tris (hydroxymethyl) aminomethane hydrochloride</td>
			<td>Himedia</td>
			<td>MB029</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Tris-acetate-EDTA</td>
			<td>SERVA</td>
			<td>42549.1</td>
			<td>Buffer /Reagent preparation</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>For cell count</td>
		</tr>
		<tr>
			<td>XhoI</td>
			<td>BioLabs</td>
			<td>R0146S</td>
			<td>Restriction enzyme</td>
		</tr>
	</tbody>
</table>

immunometabolic circuits in infection for advancing host directed therapies

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.

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...

Immunometabolic Circuits in Infection for Advancing Host Directed Therapies

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.

Immuno-metabolism is a pivotal determinant in the progression of leishmaniasis. Synthetic biology-based approach has garnered significant attention as a ...

immunometabolic-circuits-infection-for-advancing-host-directed

Research

JoVE Journal

Bioengineering

292 Views.  Biotechnology Research and Innovation Council - National Centre for Cell Science (BRIC-NCCS). 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.

Video: Immunometabolic Circuits in Infection for Advancing Host Directed Therapies

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Evaluation of the Storage Stability of Extracellular Vesicles

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical development, not only their pharmaceutical activity is important but also their production needs to be evaluated. In this context, research focuses on the isolation of EVs, their characterization, and their storage. The present manuscript aims at providing a facile procedure for the assessment of the effect of different storage conditions on EVs, without genetic manipulation or specific functional assays. This makes it possible to quickly get a first impression of the stability of EVs under a given storage condition, and EVs derived from different cell sources can be compared easily. The stability measurement is based on the physicochemical parameters of the EVs (size, particle concentration, and morphology) and the preservation of the activity of their cargo. The latter is assessed by the saponin-mediated encapsulation of the enzyme beta-glucuronidase into the EVs. Glucuronidase acts as a surrogate and allows for an easy quantification via the cleavage of a fluorescent reporter molecule. The present protocol could be a tool for researchers in the search for storage conditions that optimally retain EV properties to advance EV research toward clinical application.

Here we present a readily applicable protocol to assess the storage stability of extracellular vesicles, a group of naturally occurring nanoparticles produced by cells. The vesicles are loaded with glucuronidase as a model enzyme and stored under different conditions. After storage, their physicochemical parameters and the activity of the encapsulated enzyme are evaluated.

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical ...

Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as an&#160;innovative source of such new compounds. One bottleneck in the process is typically the elucidation of their mode-of-action. We recently established isothermal microcalorimetry as part of a routine profiling pipeline. This technology allows for investigating the effect of antibiotic exposure on the total bacterial metabolic response, including processes that are decoupled from biomass formation. Importantly, bacteriostatic and bactericidal effects are easily distinguishable without any user intervention during the measurements. However, isothermal microcalorimetry is a rather new approach and applying this method to different bacterial species usually requires pre-evaluation of suitable measurement conditions. There are some reference thermograms available of certain bacteria, greatly facilitating interpretation of results. As the pool of reference data is steadily growing, we expect the methodology to have increasing impact in the future and expect it to allow for in-depth fingerprint analyses enabling the differentiation of antibiotic classes.

The elucidation of the mode-of-action of a novel antibiotic is a challenging task in the drug discovery process. The goal of the method described here is the application of isothermal microcalorimetry using calScreener in antibacterial profiling to provide additional insight into drug-microbe interactions.

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as ...

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

Cell-Free Protein Synthesis from Exonuclease-Deficient Cellular Extracts Utilizing Linear DNA Templates

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA templates for CFPS will further enable the technology to reach its full potential, decreasing the experimental time by eliminating the steps of cloning, transformation, and plasmid extraction. Linear DNA can be rapidly and easily amplified by PCR to obtain high concentrations of the template, avoiding potential in vivo expression toxicity. However, linear DNA templates are rapidly degraded by exonucleases that are naturally present in the cell extracts. There are several strategies that have been proposed to tackle this problem, such as adding nuclease inhibitors or chemical modification of linear DNA ends for protection. All these strategies cost extra time and resources and are yet to obtain near-plasmid levels of protein expression. A detailed protocol for an alternative strategy is presented here for using linear DNA templates for CFPS. By using cell extracts from exonuclease-deficient knockout cells, linear DNA templates remain intact without requiring any end-modifications. We present the preparation steps of cell lysate from Escherichia coli BL21 Rosetta2 &#916;recBCD strain by sonication lysis and buffer calibration for Mg-glutamate (Mg-glu) and K-glutamate (K-glu) specifically for linear DNA. This method is able to achieve protein expression levels comparable to that from plasmid DNA in E. coli CFPS.

Presented here is a protocol for the preparation and buffer calibration of cell extracts from exonuclease V knockout strains of Escherichia coli BL21 Rosetta2 (&#916;recBCD and &#916;recB). This is a fast, easy, and direct approach for expression in cell-free protein synthesis systems using linear DNA templates.

Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA ...