Cell-Free Expression of Immobilized DNA

An important advantage of the DiaCHIP is the possibility to ship and store information encoded by DNA. From a DNA template array protein arrays can be produced on demand. In order to obtain this template, DNA is fixed on a silicone slide forming one side of our microfluidic chamber. Making use of a cell-free expression system, the DNA can then be transcribed and translated into the respective proteins resulting in the final protein array. The coding sequence of the proteins is genetically fused to a tag allowing their binding to a specific surface on the opposite side of the chamber.

Successful binding of DNA to the Silicone Slide

Figure 1: Scheme of the APTES/PDITC surface. The PDMS (red) is layered with APTES (light grey) and the amino-linker PDITC (dark grey).The hydroxy layer generated during plasma activation is represented in blue.

The core component of the DiaCHIP is the microfluidic chamber composed of a glass slide for protein immobilization and a PDMS (polydimethylsiloxane) flow cell. The silicone PDMS needs to be activated to enable the generation of the DNA template array by binding of the respective sequences. Oxygen plasma is used to first activate the surface of the PDMS slide by generating a hydroxy layer. This allows linking the silane APTES and finally the crosslinker PDITC. In order to bind the DNA covalently to this surface the respective DNA sequence requires an N- or C-terminal amino group. The structure of this surface is schematically visualized in figure 1. This surface chemistry is identical to the protocol used to immobilize proteins on a glass slide unspecifically (see PDITC surface for immobilizing proteins).
In order to obtain an expression cassette for GFP with an amino group, the target sequence was amplified by PCR using an amino-labeled reverse primer. Additionally, the forward primer was labeled by the fluorescent dye Cy3 to enable visualization by fluorescence microscopy. Successful amplification of the target sequence was verified by agarose gel electrophoresis.

Figure 2: Immobilization of DNA on a PDMS slide. A: Top view on the slide indicating the spotting pattern. B: Cy3 fluorescence indicating successful immobilization of amino-labeled DNA. As a negative control, Cy3- but not amino-labeled DNA was spotted.

Coupling of DNA to the PDMS slide was achieved using a DNA concentration of 25 ng/µl. Either 1 or 3 µl of DNA were spotted directly onto the slide using a distinct pattern (figure 2A). To verify that only the amino-labeled DNA specifically for amino-labeled DNA, we used non-amino-labeled DNA as a negative control and spotted the same amount. The slide was subsequently incubated over night and the DNA solution was dried afterwards at 60°C. After washing the slide, immobilized DNA was visualized by fluorescence microscopy. The resulting fluorescence pattern clearly corresponds to the spotting pattern on the slide, thereby confirming that the immobilization of DNA was successful. Spots that were incubated with amino-labeled DNA show a distinct Cy3 fluorescence signal, whereas DNA that was not labeled with an amino group was not bound to the surface (figure 2B).

Cell-Free Expression of GFP From Spotted DNA

Figure 3: GFP expressed from immobilized DNA. Fluorescence microscopy was used to visualize GFP after expression of immobilized DNA. Expression was performed for 2 h using our DiaMIX and immobilization of the His-tagged GFP was obtained by our Ni-NTA surface.

In addition to DNA immobilization itself, it is important to show that the immobilized sequence is still capable of being transcribed and translated. Therefore, the sequence was designed to contain all important elements for protein expression as promoter, ribosomal binding site and terminator. Additionally, it was adapted to the requirements of a cell-free expression system.
The microfluidic chamber consisting of the PDMS slide and an activated glass slide was assembled after immobilization of a GFP expression cassette. Using the microfluidic system, the DiaCHIP was flushed with our DiaMIX enabling cell-free expression to take place. To achieve a high yield of expressed GFP, the expression was performed for 2 h at room temperature. The glass slide was covered with our specific Ni-NTA surface. Thus, only His-tagged GFP is immobilized on the opposite site of the chamber, while other components of the DiaMIX are washed away after the expression.
The glass slide was analyzed by fluorescence microscopy afterwards. As it is shown in figure 3, cell-free expressed GFP was imobilized in distinct spots and exhibited a fluorescence signal. Thus, besides being expressed, the protein was also correctly folded and remains functional.

All in all, we showed that:

1. DNA can be immobilized reliably on a PDMS surface using an amino-label.
2. our DNA sequences are capable of being expressed by the DiaMIX although being immobilized.
3. the DiaMIX succeeds in the expression fully funtional GFP.