S175Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S165–S176

able to distinguish the dried ginseng rootlet from taproot samples andcommercial products accurately.

References

1. Dou, D.Q., Ren J., Chen Y., Pei Y.P., and Chen Y.J.: Study on the chemical constituentsof the roots of commercial ginseng. China J. Chinese Materia Medica, 28, 522-524(2003).

2. Brereton, R.G. and Chemometrics: Data Analysis for the Laboratory and ChemicalPlant. Wiley, Chichester (2003).

doi:10.1016/j.jbiosc.2009.08.474

SB-P18

Single-cell time course analysis revealed the cell divisiondependence of gene transfer

Kazumi Hakamada, and Jun Miyake

University of Tokyo, Tokyo, Japan

Non-viral gene delivery is one of the most important methods ofgene therapy. Due to the multiple extracellular and intracellularbarriers, the transfection efficacy of non-viral gene delivery has notbeen able to achieve that of viral gene delivery. Those barriers, forexample, are efficacy of endocytosis of plasmid, to escape from theendocytotic vesicle, transportation of plasmid toward the nucleus, andnuclear uptake of plasmid. Many researchers suggested that thenuclear uptake of plasmid is one of the limiting steps of the efficacy oftransfection and they had evaluated the efficacy of transfection byusing luciferase assay. Although their reports accelerated ourunderstanding of the mechanism of transfection, it is impossible toobserve the onset timing of gene expression of each cell by luciferaseassay. By clearly elucidating the rate limiting steps of transfection, thepromising new methods will be developed by logically.In this study,using HeLa cell and YFP, which was under controlled by the CMVpromoter, we evaluated transfection efficiency by using single-celltime course analysis. HeLa cell was transfected with YFP by nine non-viral transfection reagents then both phase-contrast image andfluorescent image was taken then we examined morphology of cellsand the onset timing of gene expression.

The result of all transfection reagents showed there is strongcorrelation between onset timing of gene expression and cell division.Only 1.98% of cells started to express transfected gene before celldivision. Our result revealed the non-viral vector was mainlytransported into the nucleus when the cell division and was notexpected to transport into nuclear by using other mechanisms in thenine non-viral transfection reagents.

doi:10.1016/j.jbiosc.2009.08.475

SB-P19

A systematic approach for high-level extracellularproduction of recombinant proteins in Escherichia coli

Zhi-Gang Qian,1 Xiao-Xia Xia,1 and Sang Yup Lee1,2

Metabolic and Biomolecular Engineering National Research Laboratory,Department of Chemical and Biomolecular Engineering (BK21 Program),

BioProcess Engineering Research Center, Center for Systems and Sy,Daejeon, Republic of Korea1 and Department of Bio and Brain Engineering,Department of Biological Sciences and Bioinformatics Research Center,Daejeon, Republic of Korea2

Extracellular production of recombinant proteins in Escherichia colihas received considerable attention due to its significant advantagesover cytoplasmic or periplasmic production [1]. However, efficientsecretion of recombinant proteins into the culture medium of E. coliremains a challenge due to the intrinsic limitations of the secretionmachinery. Here we report a systematic proteome-based approach forhigh-level extracellular production of recombinant proteins. First, theextracellular proteome of an E. coli B strain was analyzed to identifymotifs as potential fusion partners. Next, we expressed each openreading frame of the selected motifs and determined the proteinprofiles of the culturemedium. The highest secretingmotifwas used asthe carrier protein to produce several “passenger proteins” in theculture medium. Those model passenger proteins show a wideversatility with respect to the proteins' length and origin. In addition,the polypeptides accumulated in the medium at high concentrationsranging from 15 to 500mg/L. These findings demonstrate a proteome-based approach for high-level production of recombinant proteins inthe culture medium of E. coli [2].

Acknowledgements

This work was supported by the Korean Systems Biology ResearchProject (M10309020000-03B5002-00000) of the Ministry of Educa-tion, Science and Technology (MEST). Further supports by the WorldClass University Program from the MEST, LG Chem Chair Professor-ship, Microsoft, and IBM SUR program are appreciated.

References

1. Choi, J.H., and Lee, S.Y.: Secretory and extracellular production of recombinantproteins using Escherichia coli, Appl. Microbiol. Biotechnol., 64, 625-635 (2004).

2. Qian, Z.G., Xia, X.X., Choi, J.H., and Lee, S.Y.: Proteome-based identification of fusionpartner for high-level extracellular production of recombinant proteins in Escherichiacoli, Biotechnol. Bioeng., 101, 587-601 (2008).

doi:10.1016/j.jbiosc.2009.08.476

SB-P20

In silicoperturbationof the effects ofH2 andCO2 on themetabolismof a capnophilic bacterium, Mannheimiasucciniciproducens

Tae Yong Kim,1,2 Hyun Uk Kim,1,2 and Sang Yup Lee1,3

Metabolic and Biomolecular Engineering National Research Laboratory,Department of Chemical and Biomolecular Engineering (BK21 Pro-gram), KAIST, Daejeon, Republic of Korea1 Center for Systems andSynthetic Biotechnology, and Institute for the BioCentury, KAIST,Daejeon, Republic of Korea2 and Department of Bio and BrainEngineering, and Bioinformatics Research Center, KAIST, Daejeon,Republic of Korea3

This research presents the organism behavior of Mannheimiasucciniciproducens, a capnophilic bacterium and an efficient succinicacid producer, under varying rumen gas conditions where H2 and CO2

play a key role in production of succinic acid. Flux balance analysis ofthe genome-scale metabolic model of M. succiniciproducens was

S176 Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S165–S176

employed to estimate production patterns of several organic acids inresponse to varying influences of H2 and CO2 uptake rates. Resultsfrom controlled cultivations performed previously and constraints-based flux analyses of M. succiniciproducens in this study revealedthat there is an optimal range of CO2 level in the medium for theincrement in the cell growth rate and succinic acid production, givena glucose uptake rate. Predictions made in this study quantitativelydescribe the physiological changes of the cell in response toenvironmental perturbation, enhancing our understanding of theorganism.

Acknowledgements

This work was supported by the Genome-based IntegratedBioprocess Project of the Ministry of Education, Science and

Technology (No. 2005-01294) through the Korea Science andEngineering Foundation (KOSEF). Further supports by the LG ChemChair Professorship and by the KOSEF through the Center forUltramicrochemical Process Systems are appreciated.

References

1. Kim, T.Y., Kim, H.U., Song, H., and and Lee, S.Y.: In silico analysis of the effectsof H2 and CO2 on the metabolism of a capnophilic bacterium Mannheimiasucciniciproducens, J. Biotechnol., (in press). doi:10.1016/j.jbiotec.2009.06.003.

2. Song, H., Lee, J. W., Choi, S., You, J. K., Hong, W. H., and Lee, S. Y., Effects of dissolvedCO2 levels on the growth of Mannheimia succiniciproducens and succinic acidproduction. Biotechnol. Bioeng., 98, 1296–1304 (2007).

doi:10.1016/j.jbiosc.2009.08.477