JoVE Logo
Faculty Resource Center

Sign In





Representative Results






Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Published: October 25th, 2018



1Physics Department, Technical University of Munich
* These authors contributed equally

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.

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.

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....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Two-step lithography: Figure 5 shows the result of a two-step lithographic process on a glass slide with overlapping patterns of fluorescently labeled DIS strands.

Expression of a fluorescent protein from a gene brush: Figure 6 demonstrates the expression of the fluorescent protein YPet from immobilized DNA. At several points in time we assessed the gene expression rate.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

We gratefully acknowledge financial support for this project by the Volkswagen Stiftung (grant no. 89 883) and the European Research Council (grant agreement no. 694410 - AEDNA). M.S.-S. acknowledges support by the DFG through GRK 2062.


Log in or to access full content. Learn more about your institution’s access to JoVE content here

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
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

  1. Heyman, Y., Buxboim, A., Wolf, S. G., Daube, S. S., Bar-Ziv, R. H. Cell-free protein synthesis and assembly on a biochip. Nature Nanotechnology. 7 (6), 374-378 (2012).
  2. Jaehme, M., Michel, H. Evaluation of cell-free protein synthesis for the crystallization of membrane proteins - a case study on a member of the glutamate transporter family from Staphylothermus marinus. The FEBS Journal. 280 (4), 1112-1125 (2013).
  3. Nirenberg, M. W., Matthaei, J. H. The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proceedings Of The National Academy Of Sciences Of The United States Of America. 47, 1588-1602 (1961).
  4. Harris, D. C., Jewett, M. C. Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry. Current Opinion in Biotechnology. 23 (5), 672-678 (2012).
  5. Hodgman, C. E., Jewett, M. C. Cell-free synthetic biology: Thinking outside the cell. Metabolic Engineering. 14 (3), 261-269 (2012).
  6. Smith, M. T., Wilding, K. M., Hunt, J. M., Bennett, A. M., Bundy, B. C. The emerging age of cell-free synthetic biology. FEBS Letters. 588 (17), 2755-2761 (2014).
  7. Estévez-Torres, A., et al. Sequence-independent and reversible photocontrol of transcription/expression systems using a photosensitive nucleic acid binder. Proceedings of the National Academy of Sciences of the United States of America. 106 (30), 12219-12223 (2009).
  8. Opgenorth, P. H., Korman, T. P., Bowie, J. U. A synthetic biochemistry molecular purge valve module that maintains redox balance. Nature Communications. 5 (1), 4113 (2014).
  9. Sun, Z. Z., et al. Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology. Journal of Visualized Experiments. (79), e50762 (2013).
  10. Karzbrun, E., Shin, J., Bar-Ziv, R. H., Noireaux, V. Coarse-Grained Dynamics of Protein Synthesis in a Cell-Free System. Physical Review Letters. 106 (4), 048104 (2011).
  11. Shin, J., Jardine, P., Noireaux, V. Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-free reaction. ACS Synthetic Biology. 1 (9), 408-413 (2012).
  12. Maeda, Y. T., et al. Assembly of MreB filaments on liposome membranes: a synthetic biology approach. ACS Synthetic Biology. 1 (2), 53-59 (2012).
  13. Shin, J., Noireaux, V. An E. coli Cell-Free Expression Toolbox: Application to Synthetic Gene Circuits and Artificial Cells. ACS Synthetic Biology. 1 (1), 29-41 (2012).
  14. Niederholtmeyer, H., et al. Rapid cell-free forward engineering of novel genetic ring oscillators. eLife. 4, e09771 (2015).
  15. Shimizu, Y., et al. Cell-free translation reconstituted with purified components. Nature Biotechnology. 19 (8), 751-755 (2001).
  16. Shimizu, Y., Kanamori, T., Ueda, T. Protein synthesis by pure translation systems. Methods. 36 (3), 299-304 (2005).
  17. Pohorille, A., Deamer, D. Artificial cells: prospects for biotechnology. Trends In Biotechnology. 20 (3), 123-128 (2002).
  18. Adamala, K. P., Martin-Alarcon, D. A., Guthrie-Honea, K. R., Boyden, E. S. Engineering genetic circuit interactions within and between synthetic minimal cells. Nature Chemistry. 9 (5), 431-439 (2017).
  19. Tan, C., Saurabh, S., Bruchez, M. P., Schwartz, R., Leduc, P. Molecular crowding shapes gene expression in synthetic cellular nanosystems. Nature Nanotechnology. 8 (8), 602-608 (2013).
  20. van Nies, P., et al. Self-replication of DNA by its encoded proteins in liposome-based synthetic cells. Nature Communications. 9 (1), 1583 (2018).
  21. Buxboim, A., et al. A single-step photolithographic interface for cell-free gene expression and active biochips. Small. 3 (3), 500-510 (2007).
  22. Bracha, D., Karzbrun, E., Daube, S. S., Bar-Ziv, R. H. Emergent properties of dense DNA phases toward artificial biosystems on a surface. Accounts Of Chemical Research. 47 (6), 1912-1921 (2014).
  23. Bracha, D., Bar-Ziv, R. H. Dendritic and nanowire assemblies of condensed DNA polymer brushes. Journal of the American Chemical Society. 136 (13), 4945-4953 (2014).
  24. Daube, S. S., Bracha, D., Buxboim, A., Bar-Ziv, R. H. Compartmentalization by directional gene expression. Proceedings Of The National Academy Of Sciences Of The United States Of America. 107 (7), 2836-2841 (2010).
  25. Karzbrun, E., Tayar, A. M., Noireaux, V., Bar-Ziv, R. H. Programmable on-chip DNA compartments as artificial cells. Science. 345 (6198), 829-832 (2014).
  26. Tayar, A. M., Karzbrun, E., Noireaux, V., Bar-Ziv, R. H. Propagating gene expression fronts in a one-dimensional coupled system of artificial cells. Nature Physics. 11 (12), 1037-1041 (2015).
  27. Pardatscher, G., Schwarz-Schilling, M., Daube, S. S., Bar-Ziv, R. H., Simmel, F. C. Gene Expression on DNA Biochips Patterned with Strand-Displacement Lithography. Angewandte Chemie-International Edition In English. 57 (17), 4783-4786 (2018).
  28. Huang, F., Xu, H., Tan, W., Liang, H. Multicolor and Erasable DNA Photolithography. ACS Nano. 8 (7), 6849-6855 (2014).
  29. Huang, F., Zhou, X., Yao, D., Xiao, S., Liang, H. DNA Polymer Brush Patterning through Photocontrollable Surface-Initiated DNA Hybridization Chain Reaction. Small. 11 (43), 5800-5806 (2015).
  30. McDonald, J. C., et al. Fabrication of microfluidic systems in poly(dimethylsiloxane). Electrophoresis. 21 (1), 27-40 (2000).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved