[Frontiers in Bioscience 17, 1931-1939, January 1, 2012]

Application of cell-free expression of GFP for evaluation of microsystems

Takahiko Nojima1, Shohei Kaneda2, Hiroshi Kimura2, Takatoki Yamamoto3, Teruo Fujii2

1College of Liberal Arts and Sciences, Kitasato University, Kanagawa 252-0373, Japan, 2Institute of Industrial Science, University of Tokyo, Tokyo 153-8505, Japan, 3Department of Mechanical and Cntrol Engineering, Tokyo Institute of Technology, Tokyo 152-8550, Japan


Figure 1. PCR-based 2-input biochemical logic gates for AND, OR, NOT and AND-NOT operations. Each logic gate is wired to a single-input REPORT gate in which CFTT of GFP undergoes. A, B and C represent the PCR primers or the sequences in the template DNA. A', B' and C' represent the complement sequences to A, B and C, respectively. (A) AND gate. When both primer A and primer B are inputted, PCR undergoes, the logic gate outputs a positive signal, and the REPORT gate shows green fluorescence. (B) OR gate. Primer C is pre-mixed so that PCR progresses when A, B, or both is inputted. (C) NOT gate. B' is pre-mixed so that PCR progresses without any addition of primers, but quenched when B is inputted due to hybridization of B and B'. There are 3-nucleotides mismatch between the B-region of the template and primer B', so that the pre-mixed primer B' prefers to from a duplex with primer B when it is added. (D) AND-NOT gate. Primer C is premixed. PCR of the GFP-coding region progresses when A is inputted, but not when A+B are inputted; extensions of primers A and C are interrupted by the binding of B on the template.

Figure 2. Fluorescence of the cell-free synthesized GFP in PDMS-based microwells. The positions of each well correspond to Table 1.

Figure 3. Normalized output signals from the logic gates reported by CFTT of GFP. Measured fluorescence intensities (F) are normalized to the values obtained in the absence of input primers and template DNA (F0). A universal threshold of F/F0 = 10 was chosen to define ON (F/F0 > 10, dark bars) and OFF (F/F0 < 10, light bars) states of the logic gates. X-axis represents types of logic operation; AND, OR, NOT, and AND-NOT operations. Y-axis represents the combination of input primer; "0" and "1" mean "no input" and "input", respectively.

Figure 4. Construction of a template DNA for CFTT by using on-chip CE-based DNA collection.

Figure 5. (A) Design of the microfluidic device for the on-chip CE-based DNA collection. The size is described in mm scale. (B) Structure of the PDMS fluidic chip. (C) Layout of ports and channels. The volume of each port is less than 5 L. The channel width is 90 m and the channel depth is 30 m. The channel was filled with 1.2 % hydroxyethyl cellulose in 1xTBE buffer. The glass substrate patterned with electrodes (A) and PDMS chip (B) were brought into contact. Sample is loaded from port A to port E, and the loaded sample is separated by an electrophoresis to port C, then the target band was separated at BD-FC intersection by switching the direction of the electrophoresis. The band is collected from port B.

Figure 6. Separation and collection of the target DNA with on-chip CE-based DNA collection. (1, 2) Sample DNA mixture is loaded from Port A to Port E. (3-5) The loaded sample is separated by an electrophoresis to Port C. (6, 7) The target band is separated at the BD-FC intersection by switching the direction of electrophoresis.

Figure 7. Captured video images of the target DNA collection at the BD-FC intersection of Figure 5. The reaction mixture was stained with SYBR Green I and a 4 L portion (containing 100 ng DNA mixture) was subjected for the separation. (1, 2) The target fraction came down to the intersection in vertical direction. (3, 4) The direction of electrophoresis was switched to horizontal and the target fraction goes to the recovery port. Electrophoretic operations were implemented by a computer-controlled high voltage supply. The operation was monitored with an inverted fluorescence microscope equipped with a CCD camera and recorded by a video recorder.

Figure 8. Fluorescence of GFPuv synthesized in CFTT. DNA collected from the on-chip CE was subjected to a PCR and 1 g of the amplified DNA was subjected to a 100 L scale of a CFTT system as a template. The reaction was carried out at 30 C for 4 h. Picture was taken on a standard UV-visible transilluminator.