Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Summary

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Abstract

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

Introduction

Cell-free gene expression reactions are of great interest for various applications in biochemistry, biotechnology, and synthetic biology. Cell-free expression of proteins was instrumental for the preparation of pure protein samples, which were the basis for numerous studies in structural biology. For instance, cell-free systems were successfully used for the expression of protein complexes¹ or membrane proteins², which are difficult to produce using cell-based expression. Notably, cell-free gene expression reactions were also used to elucidate the structure of the genetic code, starting with the groundbreaking experiment....

Protocol

NOTE: A time schedule for the steps in the different sections is given in the supplementary information (section 1).

1. Chip Fabrication

NOTE: As substrates, use silicon wafers (100 mm diameter, 0.525 mm thickness) with a 50 nm thick layer of silicon dioxide or glass slides (24 mm x 24 mm, no. 1.5; 22 mm x 50 mm, no. 4). Depending on the application, other sizes and thicknesses may be more suitable.

1. Cleaning the substrates via an RCA clean proce.......

Discussion

Bephore lithography is a robust and versatile technique for the patterned immobilization of DNA or RNA. Yet, the procedure includes several steps, which - if changed - may be a source for failure or reduced performance of the system.

A crucial step in the fabrication of Bephore chips is the PEGylation of the substrate, which provides the biocompatibility of the surface. Here, the cleaning step with an RCA procedure is important, since it also activates the surface for the subsequent silanizati.......

Materials

Name	Company	Catalog Number	Comments
Silicon wafer with 50 nm silicon dioxide (Bephore substrate)	Siegert Wafer		Thickness (µm): 525 ±25, Diameter (mm): 100
Silicon wafer (for PDMS master mold)	Siegert Wafer		Thickness (µm): 525 ±25, Diameter (mm): 76.2 (3”)
Glass slides no. 4	Menzel		22 mm x 50 mm
Glass slides no. 1.5	Assistent		24 mm x 24 mm
Biotin-PEG-Silane	Laysan Bio		MW 5,000
Anhydrous toluene	Sigma Aldrich (Merck)	244511
Streptavidin	Thermo-Fisher Scientific	S888
DNA	Integrated DNA Technologies (IDT)
Phusion High-Fidelity PCR Master Mix with HF Buffer	New England Biolabs	M0531S	PCR kit
Wizard SV Gel and PCR Clean-Up System	Promega	A9281	Spin-column PCR clean-up kit
PURExpress	New England Biolabs	E6800S	Cell-free expression system
PDMS	Dow Corning	Slygard 184
FluoSpheres	Thermo-Fisher Scientific	F8771
PTFE tubing (ID: 0.8mm, OD: 1.6 mm)	Bola	S 1810-10
EpoCore 20	micro resist technology GmbH		Photoresist
mr-Dev 600	micro resist technology GmbH		Photoresist developer
Ti-Prime	MicroChemicals		Adhesion promoter
Two-component silicon glue	Picodent	Twinsil
UV-protection yellow foil	Lithoprotect (via MicroChemicals)	Y520E212
Equipment
Masks for photolithography	Zitzmann GmbH		64.000 dpi, 180x240 mm
Upright microscope	Olympus	BX51	Photolithography and fluorescence imaging
60x water immersion objective	Olympus	LumPlanFl	Used with Olympus BX51, NA 0.9
20x water immersion objective	Olympus	LumPlanFl	Used with Olympus BX51, NA 0.5
Camera	Photometrics	Coolsnap HQ	Used with Olympus BX51
Ligtht source	EXFO	X-Cite 120Q	Used with Olympus BX51
Inverted microscope	Nikon	Ti2-E	Fluorescence imaging of gene expression
4x objective	Nikon	CFI P-Apo 4x Lambda	Used with Nikon Ti2-E
Camera	Andor	Neo5.5	Used with Nikon Ti2-E
Light source	Lumencor	SOLA SM II	Used with Nikon Ti2-E
Cage incubator	Okolab	bold line	Used with Nikon Ti2-E
Pressure Controller	Elveflow	OB1 MK3
NanoPhotometer	Implen		DNA concentration measurement
Plasma cleaner	Diener	Femto	200 W, operated at 0.8 mbar with the sample in a Faraday cage