Rapid Development of Cell State Identification Circuits with Poly-Transfection

Summary

Complex genetic circuits are time-consuming to design, test, and optimize. To facilitate this process, mammalian cells are transfected in a way that allows the testing of multiple stoichiometries of circuit components in a single well. This protocol outlines the steps for experimental planning, transfection, and data analysis.

Abstract

Mammalian genetic circuits have demonstrated the potential to sense and treat a wide range of disease states, but optimization of the levels of circuit components remains challenging and labor-intensive. To accelerate this process, our lab developed poly-transfection, a high-throughput extension of traditional mammalian transfection. In poly-transfection, each cell in the transfected population essentially performs a different experiment, testing the behavior of the circuit at different DNA copy numbers and allowing users to analyze a large number of stoichiometries in a single-pot reaction. So far, poly-transfections that optimize ratios of three-component circuits in a single well of cells have been demonstrated; in principle, the same method can be used for the development of even larger circuits. Poly-transfection results can be easily applied to find optimal ratios of DNA to co-transfect for transient circuits or to choose expression levels for circuit components for the generation of stable cell lines.

Here, we demonstrate the use of poly-transfection to optimize a three-component circuit. The protocol begins with experimental design principles and explains how poly-transfection builds upon traditional co-transfection methods. Next, poly-transfection of cells is carried out and followed by flow cytometry a few days later. Finally, the data is analyzed by examining slices of the single-cell flow cytometry data that correspond to subsets of cells with certain component ratios. In the lab, poly-transfection has been used to optimize cell classifiers, feedback and feedforward controllers, bistable motifs, and many more. This simple but powerful method speeds up design cycles for complex genetic circuits in mammalian cells.

Introduction

The field of mammalian synthetic biology has rapidly progressed, from developing simple sense-and-respond parts in cultured cell lines to the optimization of complex networks of genes to address real-world challenges in diagnostics and therapeutics¹. These sophisticated circuits are capable of sensing biological inputs from microRNA profiles to cytokines to small molecule drugs, and implementing logic processing circuits including transistors, band-pass filters, toggle switches, and oscillators. They have also shown promising results in animal models of diseases like cancer, arthritis, diabetes, and many more^1,

Protocol

NOTE: Table 1 and Table 2 serve as significant references for this protocol. Table 1 shows reagent scaling for reactions, and Table 2 shows DNA ratio arithmetic for an example poly-transfection described in the protocol (upper half) and for a possible follow-up experiment (lower half).

1. Preparing cells for transfection

1. Ensure that the culture of human embryonic kidney (HEK293) cells is 60%-80% confluent before initiating the protocol. To do this, seed 1 x 10⁶ cells in a 100 mm x 15 mm tissue culture Petri dish 2 days prior, and incu....

Results

In Figure 1, we compare co-transfection to poly-transfection. In a co-transfection, all plasmids are delivered in the same transfection mix, resulting in high correlation between the amount of each plasmid any single cell receives (Figure 1A). While the number of total plasmids delivered to each cell varies significantly, the fluorescence of the two reporter proteins in the individual cells across the population is well-correlated, indicating that the two plasmi.......

Discussion

Rapid prototyping methods such as computer-aided design (CAD), breadboarding, and 3D printing have revolutionized mechanical, electrical, and civil engineering disciplines. The ability to quickly search through many possible solutions to a given challenge greatly accelerates progress in a field. We believe that poly-transfection is an analogous technology for biological engineering, enabling rapid prototyping of genetic circuits. Additionally, other rapid prototyping technologies require hands-on sequential iteration of .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15mL Corning Falcon conical tubes	ThermoFisher Scientific	14-959-53A
24-well petri dish	Any company of choice		(Non-pyrogenic, Sterile, RNase, DNase, DNA and Pyrogen Free)
Bovine serum albumin	NEB	B9000S
Centrifuge	Any company of choice		Capable of exposing 15mL Falcon tubes to 300 rcf
Countess 3 Automated Cell Counter	ThermoFisher Scientific	AMQAX2000
Countess Cell Counting Chamber Slides	ThermoFisher Scientific	C10228
Cytoflow	Non-commercial software package		https://cytoflow.readthedocs.io/en/stable/#
DMEM	VWR	10-013-CV	Use the correct media for your cell type
EDTA	ThermoFisher Scientific	03690-100ML
Fetal bovine serum	Sigma Aldrich	F4135
HEK cells	ATCC	CRL-1573	Use the relevant cell type for your experiments. HEK cells tend to transfect very efficiently.
HeLa cells	ATCC	CRL-12401	Use the relevant cell type for your experiments.
Lipofectamine 3000 and P3000 enhancer	ThermoFisher Scientific	L3000001	Use the correct reagent for your cell type; transfection and enhancer reagent
LSRFortessa flow cytometer	BD Biosciences	N/A
MEM Non-Essential Amino Acids Solution	Gibco	11140050
Microcentrifuge Tubes, 1.5 mL	Any company of choice
Opti-MEM	ThermoFisher Scientific	31985070	reduced serum medium
Phosphate buffered saline	ThermoFisher Scientific	70011044
Rainbow calibration beads	Spherotech	URCP-100-2H
Sodium azide	Sigma Aldrich	S2002
Trypsin	VWR	25-053-CI