Rapid Optimization of a Light-Inducible System to Control Mammalian Gene Expression

Summary

This study describes a method for high-throughput experiments using a 3D-printed LED array to optimize light-inducible gene expression in HEK293T cells.

Abstract

Inducible gene expression tools can open novel applications in human health and biotechnology, but current options are often expensive, difficult to reverse, and have undesirable off-target effects. Optogenetic systems use light-responsive proteins to control the activity of regulators such that expression is controlled with the "flip of a switch". This study optimizes a simplified light activated CRISPR effector (2pLACE) system, which provides tunable, reversible, and precise control of mammalian gene expression. The OptoPlate-96 enables high-throughput screening via flow cytometry for single-cell analysis and rapid optimization of 2pLACE. This study demonstrates how to use the 2pLACE system with the OptoPlate-96 in HEK293T cells to identify the optimal component ratios for maximizing dynamic range and to find the blue light intensity response curve. Similar workflows can be developed for other mammalian cells and for other optogenetic systems and wavelengths of light. These advancements enhance the precision, scalability, and adaptability of optogenetic tools for biomanufacturing applications.

Introduction

Synthetic biology tools such as inducible gene expression systems have provided significant contributions to biological research. Their ability to regulate gene expression in a tunable, reversible, and precise manner can improve control of protein production^1,2,3,4,5,6, cell morphology^7,8,9,10,11,

Protocol

1. Plating HEK293T cells in a 96-well format

1. In a biosafety cabinet, collect a bleach waste container, serological pipettes, pipette-aid, Dulbecco's Phosphate Buffered Saline (DPBS), Dulbecco-modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS), and a T-75 cell culture flask with confluent HEK293T cells. Prewarm the media to 37 °C.
2. In a T-75 flask, check that the confluency is about 80%-100% confluent through an inverted microscope.
3. In the biosafety cabinet, use a serological pipette to aspirate the spent media from the cell culture flask. Avoid touching the surface or plastic of the flask. Disp....

Results

Adopting workflow elements from previous literature^37,38,55, we added high-throughput simultaneous illumination from the OptoPlate-96 and demonstrated a pipeline to rapidly optimize a light-inducible gene expression system in mammalian cells (Figure 1).

For inducible gene expression systems, high dynamic ranges are a critical performance marker, in addition to absolute on.......

Discussion

This protocol demonstrates the optogenetic control of a multicomponent transient gene expression in mammalian cells, allowing other researchers to rapidly and simultaneously characterize modular optogenetic systems with a fluorescent reporter.

In this case, a two-plasmid light-inducible system was optimized for the expression of eGFP in HEK293T cells. For successful co-transfection, it is essential to optimize seeding densities and mass ratios for the highest transfection efficiency. This is e.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL microcentrifuge tubes	VWR	10025-724
10 mL Reagent Reservoirs	VWR	77395-252
10 mL serological pipettes	VWR	75816-100
1000 μL filter tips	VWR	76322-154
15 mL  High-Performance Centrifuge Tubes, flat cap	VWR	89039-664
1931-C Optical Power Meter	Newport	1931-C
2 mL serological pipettes	VWR	75816-104
20 μL filter tips	VWR	76322-134
200 μL filter tips	VWR	76322-150
50 mL High-Performance Centrifuge Tubes, flat cap	VWR	89039-656
96-well v-bottom plates	BRANDplates	781661
A21, LED Red light bulb	Bluex Bulbs	N/A	red light source
Arduino IDE Software	Arduino	N/A
Biosafety Cabinet, Class II	NuAire	N/A
Bright-Line Hemocytometer	Hausser Scientific	3110
Cell counting slides	BioRad	1450016
Cell Culture Plate 96-well, #1.5H glass bottom plate	Cellvis	P96-1.5H-N
CytExpert Software	Beckman Coulter	N/A
CytoFLEX Ready to Use Daily QC Fluorospheres	Beckman Coulter	C65719	4 °C
CytoFLEX-S Flow Cytometer (4 violet, 2 blue, 4 Yellow Green, 3 Red channels)	Beckman Coulter	C09766
Dulbecco's Phosphate Buffered Saline powder, no calicum, no magnesium	Fisher Scientific	21600069	Room Temperature
Eppendorf Centrifuge 5804	Eppendorf	022622501	v-bottom plate centrifuge step
Eppendorf Centrifuge 5810R	Eppendorf	22625501
Eppendorf Research plus, 8-channel, variable volume, 30 - 300 μL	Eppendorf	3125000052
Eppendorf Research plus, single-channel, variable volume, 100 - 1000 μL	Eppendorf	3123000063
Eppendorf Research plus, single-channel, variable volume, 2 - 20 μL	Eppendorf	3123000039
Eppendorf Research plus, single-channel, variable volume, 20 - 200 μL	Eppendorf	3123000055
General Purpose Laboratory Labeling Tape	VWR	89097-920
Gibco DMEM, powder, high glucose	Fisher Scientific	12100061	4 °C
Gibco Trypan Blue Solution, 0.4%	Fisher Scientific	15-250-061	Room Temperature
Gibco Value Heat Inactivated FBS	Fisher Scientific	A5256901	-20 °C
Gibco, Trypsin-EDTA (0.05%), with EDTA, Animal Origin, 1X, Phenol Red, 500 mL	Gibco	25300062	-20 °C
HEK293T Cells	ATCC	CRL-11268	-80 °C, Liquid Nitrogen
Lab markers, Micronova	VWR	89205-944
Metal Desk Lamp	Simple Designs	N/A	Lamp for red light
OptoPlate-96	LABmaker	N/A
Pipet-aid XP	Drummond	4-000-101
Plasmid: CIBN-gRNA	N/A	N/A	-20 °C
Plasmid: CMV-eGFP	N/A	N/A	-20 °C
Plasmid: CRY2-eGFP	N/A	N/A	-20 °C
PolyJet DNA In Vitro Transfection Reagent	Fisher Scientific	NC1536117	4 °C
T-75 Cell Culture Flask	VWR	10062-860
Water Jacketed CO2 Incubator	VWR	10810-884