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Abstract

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Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Published: July 6th, 2021

DOI:

10.3791/62109

1Biomedical Engineering Department, Stony Brook University, 2Laufer Center for Physical and Quantitative Biology, Stony Brook University, 3Stony Brook Medical Scientist Training Program
* These authors contributed equally

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, 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.

Similar to other engineering disciplines, synthetic biology aims to standardize protocols, allowing tools with highly reproducible functions to be utilized for exploring questions relevant to biological systems1,2. One domain in synthetic biology where many control systems have been built is the area of gene expression regulation3,4. Gene expression control can target both protein levels and variability (noise or coefficient of variation, CV = σ/µ, measured as the standard deviation over the mean), which are crucial cellular characteristics d....

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1. Gene circuit design

  1. Select genetic components to combine into a single gene circuit/plasmid (e.g., mammalian DNA integration sequence motifs32, light-responsive elements33, or functional genes34).
  2. Using any genetic engineering and/or molecular cloning software, store the DNA sequences for later use and reference, annotate each sequence, and examine all the necessary features (e.g., START codons, regulatory, or translated sequ.......

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Gene circuit assembly and stable cell line generation within this article were based on commercial, modified HEK-293 cells containing a transcriptionally active, single stable FRT site (Figure 1). The gene circuits were constructed into vectors that had FRT sites within the plasmid, allowing for the Flp-FRT integration into the HEK-293 cell genome. This approach is not limited to Flp-In cells, as FRT sites can be added to any cell line of interest anywhere in the genome using DNA editing tec.......

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Readers of this article can gain insight into the steps vital for characterizing optogenetic gene circuits (as well as other gene expression systems), including 1) gene circuit design, construction, and validation; 2) cell engineering for introducing gene circuits into stable cell lines (e.g., Flp-FRT recombination); 3) induction of the engineered cells with a light-based platform such as the LPA; 4) initial characterization of light induction assays via fluorescence microscopy; and 5) final gene expression characterizat.......

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We would like to thank Balázsi lab for comments and suggestions, Dr. Karl P. Gerhardt and Dr. Jeffrey J. Tabor for helping us construct the first LPA, and Dr. Wilfried Weber for sharing the LOV2-degron plasmids. This work was supported by the National Institutes of Health [R35 GM122561 and T32 GM008444]; The Laufer Center for Physical and Quantitative Biology; and a National Defense Science and Engineering Graduate (NDSEG) Fellowship. Funding for open access charge: NIH [R35 GM122561].

Author contributions: M.T.G. and G.B. conceived the project. M.T.G., D.C., and L.G., performed the experiments. M.T.G., D.C., L.G., and G.B. analyzed th....

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Name Company Catalog Number Comments
0.2 mL PCR tubes Eppendorf 951010006 reagent for carrying out PCR
0.25% Trypsin EDTA 1X Thermo Fisher Scientific MT25053CI reagent for splitting & harvesting mammalian cells
0.5-10 μL Adjustable Volume Pipette Eppendorf 3123000020 tool used for pipetting reactions
100-1000 μL Adjustable Volume Pipette Eppendorf 3123000039 tool used for pipetting reactions
20-200 μL Adjustable Volume Pipette Eppendorf 3123000055 tool used for pipetting reactions
2-20 μL Adjustable Volume Pipette Eppendorf 3123000039 tool used for pipetting reactions
5 mL Polystyene Round-Bottom Tube w/ Cell Strainer Cap Corning 352235 reagent for flow cytometry
5702R Centrifuge, with 4 x 100 Rotor, 15 and 50 mL Adapters, 120 V Eppendorf 22628113 equipment for mammalian culture work
Agarose Denville Scientific GR140-500 reagent for gel electrophoresis
Aluminum Foils for 96-well Plates VWR® 60941-126 tool used for covering plates in light-induction experiments
Ampicillin Sigma Aldrich A9518-5G reagent for selecting bacteria with correct plasmid
Analog vortex mixer Thermo Fisher Scientific 02215365PR tool for carrying PCR, transformation, or gel extraction reactions
Bacto Dehydrated Agar Fisher Scientific DF0140010 reagent for growing bacteria
BD LSRAria BD 656700 tool for sorting engineered cell lines into monoclonal populations
BD LSRFortessa BD 649225 tool for characterizing engineered cell lines
BSA, Bovine Serum Albumine Government Scientific Source SIGA4919-1G reagent for IF incubation buffer
Cell Culture Plate 12-well, Clear, flat-bottom w/lid, polystyrene, non-pyrogenic, standard-TC Corning 353043 plate used for growing monoclonal cells
Centrifuge VWR 22628113 instrument for mammalian cell culture
Chemical fume hood N/A N/A instrument for carrying out IF reactions
Clear Cell Culture Plate 24 well flat-bottom w/ lid BD 353047 plate used for growing monoclonal cells
CytoOne T25 filter cap TC flask USA Scientific CC7682-4825 container for growing mammalian cells
Dimethyl sulfoxide (DMSO) Fischer Scientific BP231-100 reagent used for freezing down engineered mammalian cells
Ethidium Bromide Thermo Fisher Scientific 15-585-011 reagent for gel electrophoresis
Falcon 96 Well Clear Flat Bottom TC-Treated Culture Microplate, with Lid Corning 353072 container for growing sorted monoclonal cells
FCS Express De Novo Software: N/A software for characterizing flow cytometry data
Fetal Bovine Serum, Regular, USDA 500 mL Corning 35-010-CV reagent for growing mammalian cells
Fisherbrand Petri Dishes with Clear Lid - Raised ridge; 100 x 15 mm Fisher Scientific FB0875712 equipment for growing bacteria
Gibco DMEM, High Glucose Thermo Fisher Scientific 11-965-092 reagent for growing mammalian cells
Hs00932330_m1 KRAS isoform a Taqman Gene Expression Assay Life Technologies 4331182 qPCR Probe
Hygromycin B (50 mg/mL), 20 mL Life Technologies 10687-010 reagent for selecting cells with proper gene circuit integration
iScript Reverse Transcription Supermix Bio-Rad Laboratories 1708890 reagent for converting RNA to cDNA
Laboratory Freezer -20 °C VWR 76210-392 equipment for storing experimental reagents
Laboratory Freezer -80 °C Panasonic MDF-U74VC equipment for storing experimental reagents
Laboratory Refrigerator +4 °C VWR 76359-220 equipment for storing experimental reagents
LB Broth (Lennox) , 1 kg Sigma-Aldrich L3022-250G reagent for growing bacteria
LIPOFECTAMINE 3000 Life Technologies L3000008 reagemt for transfecting gene circuits into mammalian cells
MATLAB 2019 MathWorks N/A software for analyzing experimental data
Methanol Acros Organics 413775000 reagent for immunofluorescence reaction
Microcentrifuge Tubes, Polypropylene 1.7 mL VWR 20170-333 plasticware container
Mr04097229_mr EGFP/YFP Taqman Gene Expression Assay Life Technologies 4331182 qPCR Probe
MultiTherm Shaker Benchmark Scientific H5000-HC equipment for bacterial transformation
NanoDrop Lite Spectrophotometer Thermo Fisher Scientific ND-NDL-US-CAN equipment for DNA/RNA concentration measurement
NEB Q5 High-Fidelity DNA polymerase 2x Master Mix NEB M0492S reagent for PCR of gene circuit fragments
NEB10-beta Competent E. coli (High Efficiency) New England Biolabs (NEB) C3019H bacterial cells for amplifying gene circuit of interest
NEBuilder HiFi DNA Assembly Master Mix New England Biolabs (NEB) E2621L reagent for combining gene circuit fragements
Nikon Eclipse Ti-E inverted microscope with a DS-Qi2 camera Nikon Instruments Inc. N/A instrument for quantifying gene expression
NIS-Elements Nikon Instruments Inc. N/A software for characterizing fluorescence microscopy data
oligonucleotides IDT N/A reagent used for PCR of gene circuit components
Panasonic MCO-170 AICUVHL-PA cellIQ Series CO2 Incubator with UV and H2O2 Control Panasonic MCO-170AICUVHL-PA instrument for growing mammalian cells
Paraformaldehyde, 16% Electron Microscopy Grade Electron Microscopy Sciences 15710-S reagent
PBS, Dulbecco's Phosphate-Buffered Saline (D-PBS) (1x) Invitrogen 14190144 reagent for mammalian cell culture,reagent for IF incubation buffer
Penicillin-Streptomycin (10,000 U/mL), 100x Fisher Scientific 15140-122 reagent for growing mammalian cells
primary ERK antibody Cell Signaling Technology 4370S primary ERK antibody for immunifluorescence
primary KRAS antibody Sigma-Aldrich WH0003845M1 primary KRAS antibody for immunifluorescence
QIAprep Spin Miniprep Kit (250) Qiagen 27106 reagent kit for purifying gene circuit plasmids
QIAquick Gel Extraction Kit (50) Qiagen 28704 reagent kit for purifying gene circuit fragments
QuantStudio 3 Real-Time PCR System Eppendorf A28137 equipment for qRT-PCR
Relative Quantification App Thermo Fisher Scientific N/A software for quantifying RNA/cDNA amplificaiton
RNeasy Plus Mini Kit Qiagen 74134 kit for extracting RNA of engineered mammalian cells
Secondary ERK antibody Cell Signaling Technology 8889S secondary ERK antibody for immunifluorescence
secondary KRAS antibody Invitrogen A11005 secondary KRAS antibody for immunifluorescence
Serological Pipets 5.0 mL Olympus Plastics 12-102 reagents used for setting up a variety of chemical reactions
SmartView Pro Imager System Major Science UVCI-1200 tool for imaging correct PCR bands
SnapGene Viewer (free) or SnapGene SnapGene N/A software DNA sequence design and analysis
Stage top incubator Tokai Hit INU-TIZ tool for carrying PCR, transformation, or gel extraction reactions
TaqMan Fast Advanced Master Mix Thermo Fisher Scientific 4444557 reagent for PCR of gene circuit fragments
TaqMan Human GAPD (GAPDH) Endogenous Control (VIC/MGB probe), primer limited, 2500 rxn Life Technologies 4326317E qPCR Probe
Thermocycler Bio-Rad 1851148 tool for carrying PCR, transformation, or gel extraction reactions
VisiPlate-24 Black, Black 24-well Microplate with Clear Bottom, Sterile and Tissue Culture Treated PerkinElmer 1450-605 plate used for light-induction experiments
VWR Disposable Pasteur Pipets, Glass, Borosilicate Glass Pipet, Short Tip, Capacity=2 mL, Overall Length=14.6 cm VWR 14673-010 reagent for mammalian cell culture
VWR Mini Horizontal Electrophoresis Systems, Mini10 Gel System VWR 89032-290 equipment for DNA gel electrophoresis
Flp-In 293 Thermo Fisher Scientific R75007 Engineered cell line with FRT site

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