Fabrication and Use of a 32-Well LED-Embedded Microplate for Optogenetic Dynamic Control

Summary

This protocol describes how to fabricate, program, and operate a 32-well light-emitting diode (LED)-embedded microplate for optogenetic studies (LEMOS 2.0) inside a microplate reader to enable high-throughput optogenetic stimulation and quantitative gene expression measurements in microbial cultures.

Abstract

Optogenetic control enables light-actuated regulation of gene expression and provides a programmable interface between living cells and electronic systems. However, routine prototyping of optogenetic constructs remains limited by infrastructure. Existing closed-loop platforms often require chemostats, microfluidics, robotic handling, or custom optical sensors, which can increase cost, reduce accessibility, or constrain measurement performance.

Here, LEMOS 2.0 is presented, an updated light-emitting diode (LED)-embedded microplate for optogenetic studies, a low-cost device for optogenetic stimulation and gene-circuit characterization inside standard microplate readers. LEMOS 2.0 builds on the original LEMOS platform by increasing throughput from 16 to 32 microwells and reducing light leakage between adjacent microwells, enabling dark conditions as an additional illumination state. The device consists of a three-dimensional (3D)-printed frame, individually addressable LEDs positioned next to each microwell, a rechargeable battery, and an onboard microcontroller for Bluetooth-based wireless communication. Biocompatible polydimethylsiloxane (PDMS) microwells are cast directly into the device by replica molding, enabling the stimulation of bacterial cultures through the transparent mold. The device fits into standard plate readers, enabling optical density and fluorescence measurement.

This protocol describes the full LEMOS 2.0 workflow, including device fabrication, circuit assembly, Arduino programming, PDMS microwell casting, plate-reader setup, strain and culture preparation, automated experiment execution, device cleanup, and fluorescence/OD₆₀₀ data analysis. As a demonstration, the protocol uses the CcaSR optogenetic system, in which sfGFP expression is activated by green light and repressed by red light. LEMOS 2.0 is intended to make optogenetic perturbation and gene-expression characterization more accessible to wet-lab users, enabling faster design-build-test-learn cycles without requiring specialized bioreactor or microfluidic infrastructure.

Introduction

Optogenetics provides a programmable interface between living cells and electronic control systems. By using light to regulate gene expression, cellular behavior can be actuated with temporal precision, reversibility, and minimal perturbation to the culture medium¹. When paired with fluorescent or luminescent reporters, optogenetic systems allow biological states to be measured electronically and regulated through feedback¹. This bidirectional optical interface has enabled cybergenetic control of cellular processes, including gene expression, cell growth, and population composition^2,

Protocol

All experimental procedures involving Escherichia coli strain MG1655  were performed in designated Biosafety Level 2 (BSL-2) laboratories at Texas A&M University. Bacterial cultures and manipulations were carried out in certified Class II Biosafety Cabinets in accordance with standard containment guidelines. Procedures were performed under designated containment conditions reviewed and approved by the Texas A&M Institutional Biosafety Committee (IBC) under permit number IBC2023-044.

1. Fabrication of the LEMOS 2.0 Frame and LED Array

NOTE: Begin fabricating LEMOS....

Results

LEMOS 2.0 is a 32-well, microplate-reader-compatible platform²⁴ for programmable optogenetic stimulation and quantitative gene-expression measurement (Figure 2A,B). Each microwell is paired with an individually addressable WS2812B light-emitting diode (LED), controlled by an Arduino Nano 33 BLE Sense Rev2 through the Adafruit_NeoPixel library. The device communicates with an external computer through Bluetooth, allowing illumination commands to be upd.......

Discussion

This protocol describes the fabrication and operation of LEMOS 2.0, a 32-well LED-embedded microplate for optogenetic stimulation and feedback-control experiments inside a standard microplate reader. Representative experiments demonstrate that LEMOS 2.0 supports microbial growth (Figure 2E) with no significant phototoxicity observed under illumination. Additionally, LEMOS 2.0 reduces light leakage into adjacent dark-condition microwells (Figure 3D), thereby impr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
22 AWG stranded hookup wire	Amazon	ASIN: B089CQ2N69	Inexpensive wire to connect the electrical circuit. Solid or stranded wire is acceptable.
8 Pin 3 Position 2P3T DP3T Slide Switch Side Knob	Amazon	ASIN: B007Q9Y8YC	Switch and pins should be on adjacent sides, not opposite, as is more commmon.
Arduino Nano 33 BLE Rev 2	Arduino	ABX00071	Microcontroller – Flat profile and BLE capability suits the peripheral device needs.
Arduino Nano 33 IoT	Arduino	ABX00027	Microcontroller – Used as the central device.
BioTek Synergy H1 microplate reader	Agilent (BioTek)	—	Or compatible plate reader with kinetic mode
Breathe-Easy sealing film	USA Scientific	9123-6100	Medical-grade sealing film; Gas-permeable microplate seal
Chloramphenicol (25 mg/mL stock in ethanol)	Various	—	Working conc. 25 µg/mL
E. coli MG1655
Ease Release 200	Mann Release Technologies		Release agent
Kapton Tape	Amazon	ASIN: B07F8TZZ4N
LB agar plates and appropriate antibiotics	In-house	—	It is assumed that these are already present.
Li-ion 3.7 V battery, 3000 mAh (11.1 Wh), size 104060	Ufine	UFX0391-03	Should come with JST PH 2-Pin Cable Male. Manufactured by Liter Energy, sold by various retailers.
Li-ion battery charge and discharge module with Type-C USB (5V 1A)	Amazon	ASIN: B0CTMJMRKZ	Search "Replaces TP4056" on Amazon. Scroll past the standard TP4056 modules. Stop at any listing showing the smaller, black modules that read "Replaces TP4056" in the title.
Li-ion JST PH 2-Pin Cable – Female Connector 100 mm	Various	ASIN: B07NWNPB77	Must be compatible with the battery’s JST connector.
Mini Boost Module 3.7 V to 5 V/8 V/9 V/12 V Step Up Board with LED indicator	Amazon (Teyleten Robot)	ASIN: B0B4TB9QNY	[https://a.co/d/03ASKZPq](https://a.co/d/03ASKZPq)
Nunc OmniTray Single-Well Plate	Thermo Scientific	242811	Outer Shell
pNO286-3	Addgene	Plasmid #107746	Encodes constitutive CcaS expression
pSR58.6	Addgene	Plasmid #63176	Encodes constitutive CcaR expression and sfGFP under the PcpcG2 promoter
Soldering iron and solder	Various	—	Recommendations: Fine tip, flux or rosin core solder.
Spectinomycin (50 mg/mL stock)	Various	—	Working conc. 50 µg/mL
Sterile culture tubes (14 mL round-bottom)	Various	—
SYLGARD 184 silicone elastomer (base + curing agent)		SYLGARD 184	1:10 curing\:base by mass
USB-A to Micro USB cable	Various	—	For connecting Arduino Boards to the PC. Must be a data-transfer wire, not simply a charging wire.
USB-A to USB-C Charging Cable	Various	—	Connects to the battery charging module
Vacuum desiccator and pump	Various	—	For degassing PDMS
WS2812B IC SPI RGB Pixel LED Strip, 100LED/m, DC5V, IP30, Black	Amazon (BTF-LIGHTING)	ASIN: B088B8G8LD ; BTF-LIGHTING Part No.: WS2812B1M100LB30	Individually Accessible LED Strip. 100 LED/m. Voltage: 5 V. IP30 refers to water resistance. Black is preferred over white to reflect less light.