[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


1. Abstract
2. Introduction
3. Application of cell-free GFP-expression as a report gate in biomolecular logic gate system
4. Application of cell-free GFP-expression as a reporter in on-chip capillary electrophoresis fractionation of DNA
5. Summary and perspective
6. Acknowledgements
7. References


Coupled cell-free transcription-translation (CFTT) of green fluorescent protein (GFP) has been applied as a reporter system to microfluidic chip-related technologies. In polymerase chain reaction (PCR)-based biomolecular logic gate system, in which addition of primer set and amplification of PCR product represent input and output signal respectively, GFP gene was inserted in the template DNA, which was then amplified, transcribed and translated to GFP. The green fluorescence reported as if the amplification has occurred or not, that is, the fluorescence reports positive output signal. CFTT of GFP was also adopted to evaluate on-chip capillary electrophoresis (CE)-based DNA fractionation, which was developed to isolate single DNA species from reaction mixture of DNA ligase-catalyzed DNA-assembly. As a model system, GFP gene was inserted in the target DNA fragment. The collected fraction was amplified with PCR and subjected to a CFTT system, and green fluorescence was observed showing that the fractionation was successful. These results showed that CFTT of GFP is a useful tool to verify, estimate, and monitor microfluidic chip-related technologies in which cell-free protein synthesis is involved.


Production of proteins by biological synthesis based on recombinant DNA technology has been a basic technique in modern biosciences and biotechnologies (1). A variety of proteins are synthesized based on this technology today. Host cell incorporates recombinant DNA carrying genetic information and transcribes it to messenger RNA (mRNA), which is then served as a template for the protein biosynthesis. Because recombinant DNA is introduced into living host cells, the protein synthesis should be carried out in a biohazard facility. This fact has prevented us from applying the recombinant DNA technology into other technologies including electronics, informatics, or nanotechnology. Cell-free translation (CFT), otherwise known as in vitro translation (IVT), is therefore attractive approach because it requires no living cells for protein production and thereby no usage of biohazard facilities. There are two options in the way to input the genetic information into CFT reaction according to the substances: DNA or mRNA. DNA-dependent mRNA synthesis can be carried out together with CFT by adding RNA polymerase and ribonucleoside triphosphates. It is coupled cell-free transcription-translation (CFTT). Thanks to the improvement of CFT systems (2-5), various kinds of recombinant proteins are prepared in CFT today. Most of them are CFTT system.

Recently, much attention has been paid for CFT from the viewpoint of not only protein production but also protein screening (6-8). Especially, after the completion of human genome sequencing project (9,10), demand has been raised for conversion system that converts genetic information to protein one by one. One solution for this demand is miniaturized reactor for CFT. We therefore developed a microstructure, having 0.2 �L volume, fabricated in a silicon wafer, and installed cell-free translation system prepared from E. coli on it (11,12). We demonstrated two kinds of mRNA-dependent polypeptide synthesis: polyphenylalanine (poly(Phe)) synthesis depending on polyuridylic acid (poly(U)), where 14C-labeled phenylalanine was incorporated into acid-insoluble fraction, and polymerization of 14C-labeled amino acids depending on bacteriophage MS2 mRNA It was the world's first achievement of CFT in a microfabricated structure. To confirm as if the protein synthesized in a microfabricated structure has enzymatic activity, the synthesized material should be analyzed with biochemical assay. Baba and his group compared the biochemical specificity of the proteins synthesized in a microstructure, a plastic tubes, and living cells, and showed no differences among them (13).

We introduced GFP (14,15) as a model protein synthesized in a microfabricated structure because of its convenience for detection that it shows green emission without any substances or chemical energy sources. For the observation of cell-free GFP expression, we constructed microreactor array on a transparent plastic plate, embedded on a temperature control unit (16). The reaction volume was 125 nL. We adopted two kinds of commercially available GFP variant as the first step toward multi-parallel protein expression. The variants were an ultraviolet-optimized GFPuv (F99S, M153T, V163A) (17) and a blue-shifted BFP (F64L, S65T, T145F) (18). They have same excitation wavelength, around 380 nm, so that the setting of optical equipments was compatible with standard system optimized for fluorescein detection (19). The emission wavelengths are 440 nm and 509 nm for BFP and GFPuv, respectively. The GFP synthesis by CFTT was monitored with charge coupled device (CCD) camera. We demonstrated that GFP variants were successfully synthesized in the microreactor. Parameter-tuning and system-optimization for protein synthesis in this microreactor system were carried out based on GFP-expression, and our system could translate not only GFP variants but also other proteins (20).

After that, CFTT of GFP has become a standard reaction as a reporter in the field of micro- and nano-scale biotechnology involving CFT. The reaction has been adopted in picoliter chamber array (21), nanowell chip (22), and picoliter droplets (23).

In this review, we introduce our approach to apply CFT of GFP as (i) a report gate in PCR-based biochemical logic system, and (ii) verification tool for DNA collection with on-chip capillary electrophoresis system.

The first topic in this review is PCR-based biochemical logic gate where CFT of GFP is utilized as a report gate to monitor the amplification (24). Much attention has been paid to designing a biomolecular system that responds to chemical inputs and remarkable progress has been achieved in the development of logic gates based on both proteins and nucleic acids. For example, Katz and his co-worker have reported enzyme-based logic systems for information processing, regarding enzyme's redox activity as signal processing (25). Willner and his group have developed DNA-based logic gate systems, focusing on DNA's abilities as signal processing, such as structure formation, molecular recognition, and amplification, (26). Our approach has started from the viewpoint that standard PCR (27) is 2-input AND gate. The reaction responds to the input of two kinds of primers, and DNA amplification responds to output signal. Usually, the product is analyzed with TaqMan chemistry (28), Molecular Beacon (29), or electrophoresis, however, we adopted CFTT of GFP instead of them. The positive signal is reported as green emission.

The second topic is DNA fractionation with on-chip CE, where CFTT of GFP is utilized to verify as if the target DNA fragment is collected properly (30). Although various kinds of recombinant proteins are synthesized with CFTT system, the template DNA subjected to the CFT reaction is still prepared with living cell-based method because the template is constructed with standard recombinant DNA technology, in which DNA ligase-catalyzed DNA assembly and living cell-dependent screening of the construction are utilized. Because DNA ligase assembles DNA fragments randomly to generate complex mixture (31), it is required to separate the target DNA species from the mixture, where living cell-based selection has been widely utilized, which should be carried out in a biohazard facility. In order to construct the template DNA without using living cells, we performed direct PCR from the ligation mixture, followed by collection with on-chip fractionation with CE. We adopted GFP-coding gene as a model DNA and after the fractionation, the DNA was subjected to CFTT system. Green fluorescence showed the total process was carried out properly.


Figure 1 shows PCR-based biomolecular logic gate system. This system is consisted of two reactions: one was PCR and another was CFTT. In the PCR, input signal is addition of primers, and output signal is the amplification of the target DNA. Normal PCR is 2-input AND logic gate because it undergoes responding to input of two kinds of primers. In this study, designing the templates appropriately and combining two primers or three, we constructed PCR-based AND, OR, NOT and AND-NOT logic gates. Our logic gate was a reaction mixture of PCR containing linear DNA template (logic gate template) coding GFPuv and a single stranded oligodeoxyribonucleotide (ODN) that hybridizes with logic gate template (pre-mixed primer). The logic operation is executed by an addition of primer A and/or primer B as an input signal. Since the logic gate templates carried recognition sequences for transcriptional and translational machinery, the amplified GFPuv-coding sequence is translated to GFPuv throughout CFTT, wired to the logic gates as a REPORT gate, that is, the output from the PCR-based logic gate is reported by green fluorescence.

The AND gate is a standard PCR mixture where both primer A and primer B are required to progress PCR; when both primers are inputted, the REPORT gate shows positive output signal. In the OR gate, primer C is pre-mixed in the initial condition and the logic gate operation is executed by inputting primer A, primer B, or both. In the NOT gate, primer B' is pre-mixed that carries a complement sequence to B so that the PCR undergoes without any addition of primers. When primer B is added to the reaction, the PCR is strongly inhibited due to the hybridization of B with B'. There is a 3-nucleotide mismatch between primer B' and the B-region of the template, whereas primers B and B' are perfect matched, and the molar ratio of B/B' was 5/4. Addition of B to the NOT gate therefore perfectly blocks the PCR. In the AND-NOT gate, inputting primer A progresses the PCR because C is already premixed. When both primer A and primer B are added, amplification of mature GFPuv-coding region is inhibited by formation of truncated products between A and B and between B and C.

After 50-microL scale PCR of each logic gate, 10 microL of the reaction was directly subjected to a CFTT (total volume was 50 microL) and 10 microL from it was transferred into a 10-microL microwell on a polydimethylsiloxane (PDMS)-based reaction chamber (32). The chamber was fabricated by putting a 2-mm thick PDMS-seat having two-dimensional (2-D) array of numerous micro-holes (the diameter was 3 mm) onto a flat glass substrate to form the 2-D array of microchamber (the volume was 19 �L). Each chamber was filled with the 10 �L of reaction mixture, sealed the open top with a thick polycarbonate film (thickness was 0.1 mm) to prevent evaporation during the reaction, and incubated at 30 �C for 120 min. The output signal was validated on a standard UV-visible transilluminator and photographed (Figure 2). Fourty microL-mixture left was also incubated in a tube at 30 �C, and the fluorescence intensities were quantified. Figure 3 shows the normalized intensities of output signals from the REPORT gates. These results show good agreement with theoretical truth table (Table 1) showing that all the PCR-based logic gates representing AND, OR, NOT, and AND-NOT gates properly functioned.

Adopting CFT of GFP as the single-input report gate is the characteristics in this study. The REPORT gate cuts off a background signal from the PCR-based logic gate such as non-specific priming or primer dimer formation, which gives positive signal in TaqMan or Molecular Beacon methods. For example, in NOT gate, addition of primer B quenches the PCR due to the formation of B-B' duplex, which is recognized as an output signal in DNA-level, however, the REPORT gate shows no output signal.

We are trying to expand the variation of logic operation: NAND, NOR and NOR. The construction is underway, and CFT of GFP will be adopted as the REPORT gate again.


To realize a method for DNA construction without living cell-based screening, we introduced on-chip CE fractionation of DNA construct and enzymatic reactions. The model system we employed was introduction of GFPuv gene into an expression vector carrying T7 RNA polymerase-specific promoter and terminator (33), and bacterial translational machinery. When the construction is succeeded, transcription of the target gene will be under the control of T7 RNA polymerase, and green fluorescence will observed after CFT of GFP.

The backbone of expression vector coding recognition sites for T7 RNA polymerase and bacterial translational machinery, and the insert carrying GFPuv gene were isolated from commercial vectors with a standard PCR using primer set carrying restriction sites. After the digestion with restriction enzymes, the PCR-amplified fragments were assembled with T4 DNA ligase with a standard condition (34). From the reaction mixture, the complete region that T7 RNA polymerase transcribes was amplified with PCR (1st PCR).

The amplified DNA was then collected with an on-chip CE-based DNA collection using a microfluidic device (35,36). Figure 5 illustrates design of the device for the on-chip CE-based DNA collection. Electrodes were patterned on a glass substrate, and covered with PDMS-based fluidic chip in which six ports for sample injection and collection, and electrophoretic channels were fabricated and filled with 1.2 % hydroxyethyl cellulose (37) in 1 x TBE buffer. Aliquot of PCR mixture was loaded from port A to port E, and the loaded sample was 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 operation was monitored with CCD camera coupled with a video recorder.

Figure 7 shows captured images of the fluidic operation. This operation was repeated twice and total 8 �L of sample was recovered. Without any purification, the recovered material was directly subjected to a PCR (2nd PCR) as a starting material. After the reaction, 1 �g of the amplified DNA was subjected to CFTT containing T7 RNA polymerase, and after 4 h incubation for maturing at 30 �C (38), the product eliminated green fluorescence (Figure 7). It shows that the construction of the recombinant template DNA for CFTT was successful in a cell-free manner.


In order to estimate the performance of protein synthesis reaction, it is required to monitor the activity of the protein synthesized in the system. GFP is an ideal protein for this purpose, especially for the evaluation of protein synthesis carried out in a microstructure, in which supplying substrates or sampling is difficult. In this review, we introduced our approach to apply CFTT of GFP into (i) PCR-based biomolecular logic gate system as a report gate, and (ii) on-chip CE-based DNA fractionation as a confirmation reaction. In both study, due to the feature of GFP, execution in logic gate and fractionation in on-chip CE were simply monitored.

DNA-based computing and DNA-based logic operation have been attracting interest in many field, including nanotechnology, electronics, materials, and informatics. PCR-based logic gate system is advantageous because it realizes various logic operations by designing the construction and sequences of template DNA and pre-mixed primers, without changing input primer set. We are now trying to expand the variation of the logic operation.

Because DNA is water-soluble biomolecule, and usually handled as an aqueous solution, it is of immediate interest to apply microfluidic technology to DNA-based computing and DNA-based logic operation. To build up the PCR-based logic gate system for a fluidic computing system, integration of PCR and CFTT on one chip is necessary.

Development of a platform in which total operation of gene expression including PCR and CFTT is also desired in the field of genetic engineering. Although we achieved total operation of genetic engineering in cell-free format by applying on-chip CE fractionation of DNA instead of living cell-based screening, each step is still separated. Integration and systematization of each step on one device, a useful tool would be realized for protein preparation under cell-free condition. It would open the door for "cell-free genetic engineering".

Both logic gate system and cell-free genetic engineering, we have to estimate the performance of the protein expression reaction. CFT of GFP is capable as an ideal reporter system.


This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (20550148 and 23550197) and Chuo University Joint Research Grant 2009.


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Abbreviations: CCD: charge coupled device, CE: capillary electrophoresis, CFTT: coupled cell-free transcription-translation, GFP: green fluorescent protein, mRNA: messenger ribonucleic acid, ODN: oligodeoxyribonucleotide, PCR: polymerase chain reaction, PDMS: polydimethylsiloxane

Key Words: Capillary electrophoresis, DNA, GFP, Logic gate, Microreactor, Transcription, Translation, Review

Send correspondence to: Takahiko Nojima, College of Liberal Arts and Sciences, Kitasato University, Kanagawa 252-0373, Japan, Tel: 81-42-778-8088, Fax: 81-42-778-8088, E-mail:nojima@kitasato-u.ac.jp