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erm goal is the identification of microorganisms which eitherhemselves or whose enzymes could be used as feed additives forastrointestinal fumonisin detoxification.

Previously, we isolated the fumonisin degrading alphapro-eobacterium Sphingopyxis sp. MTA144. Our present goal was theharacterization of this strain regarding its fumonisin catabolism.

We found that the hydrolysis of the mycotoxin in the clearysate of the strain was much faster when the biomass was inducedith 10 mg/l FB1 prior to the decomposition experiment itself. We

oncluded that the fum genes are not constitutively expressed, butnducible. Two genes for transcriptional regulators in the fum geneluster may be responsible for the observed gene expression regula-ion. FB1 degradation experiments with biomass of strain MTA144howed that FB1 was not only catabolised in mineral medium, butlso in complex growth medium, indicating that there is little oro catabolite repression. Moreover, we found that the presence ofp to 100 mg/l FB1 in the medium promoted growth of Sphingopy-is sp. MTA144, but had no effect on related strains which do notatabolise fumonisins. This indicates that Sphingopyxis sp. MTA144egrades FB1 for energy gain or as a carbon source rather than forelf-protecting detoxification.

ttp://dx.doi.org/10.1016/j.nbt.2012.08.466

oster 5.0.27

onstruction of specifically expressed vector in mam-ary gland for lacS and its transfection into bovine fetalbroblasts mediated by liposome

uanmin Zhou∗, Lu Li

College of Life Science, Inner Mongolia Agricultural University, Hohhot,nner Mongolia, PR China

his study was designed to optimize conditions for transfectingmammary gland specific transgene into bovine fetal fibroblasts.0.92 kb fragment of bovine �-lactoglobulin gene sequence was

btained from bovine genome by PCR amplification and insertednto the T site of pMD19-Simple T plasmid. A 1.49 kb of lacS geneoding sequence was cloned from sulfolobus solfataricus genomey PCR amplification and inserted into the pUC19 plasmid. Theoding sequence of Neor were derived by PCR amplification fromIRES2-EGFP plasmid and inserted into the BamHI/NheI site ofIRES2-EGFP plasmid. The resultant vector (pNIE) contained aeor and an EGFP gene, which were linked by an internal ribosome

ntry site (IRES) sequence downstream of the cytomegalovirusCMV) promoter. Finally, the vector pNIE was assembled into theUC19 plasmid thus creating a pBLI vector, which contained theeor and EGFP gene regulated by CMV promoter for expression innon-tissue specific mode, and the lacS gene regulated by bovine-lactoglobulin promoter for specific expression in mammaryland. 2 × 105 bovine fetal fibroblasts (bFF) cells were transfectedn DMEM with pBLI using TRANSfection for 48 h. The transfectedells were cultured for 48 h before adding G418 at concentrationsf 800 �g/mL for 14 d. The results shown that 1.0 �g pBLI plas-id and 3 �L TRANSfection yielded the desirable efficiency of

ransfection, which indicated that a specifically expressed-vectorn mammary gland for lacS gene was successfully constructed,

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168 www.elsevier.com/locate/nbt

New Biotechnology · Volume 29S · September 2012

ransfection parameters were developed and an efficient screeningeasures were established for detecting transgenic somatic cells.

ttp://dx.doi.org/10.1016/j.nbt.2012.08.467

oster 5.0.28

tructural and functional analysis of lipase gene of Ams-cta moorei entomopoxvirus

mine Ozsahin∗, Kazim Sezen, Zihni Demirbag

Karadeniz Technical University, Faculty of Science, Department of Biol-gy, Trabzon, Turkey

he entomopoxvirus isolated from the larvae of the moth, Amsactaoorei, is a distant relative of the better studied orthopoxviruseshich include variola and vaccinia virus. A. moorei ento-opoxvirus (AmEPV) has 279 unique open reading frames (ORF).mong these, AMV133 has 864 nt coding for a protein of 288mino acids. Sequence derived amino acid analysis of AMV133uggested it to be a putative triacylglyceride lipase gene, whichould conceivably function as a virulence gene through lipidydrolysis.

AMV133 was transcribed as delayed early class of temporal cas-ade since it was not inhibited by an inhibitor of DNA synthesis,owever it effected by a protein synthesis inhibitor. Transcriptiontarted 6th hours post infection and continued until 72 hpi.

5′-RACE analysis showed that transcription initiated at position77, relative to the translational start site of this gene. To deter-ine the limits of the putative promoters, upstream sequences of

arious lengths were cloned in front of a firefly luciferase reporterene. The resulting plasmid constructs were tested in a dual assay.he promoter activity was lost when the length of the sequencepstream of the translational start site was reduced from −82 to21 nucleotides.

Lipase gene of AmEPV was cloned and expressed in both bac-lovirus and bacterial expression vector systems. Purified proteinas used for lipase assay. The results show that the purified proteinas lipase activity.

Keywords: Amsacta moorei entomopoxvirus; Lipase; Transcrip-omic; Promoter analysis

ttp://dx.doi.org/10.1016/j.nbt.2012.08.468

oster 5.0.29

n LCMSMS method to analyse phenolic profile in the liq-id extract, with woodland strawberry (Fragaria vesca

.) application

amay Seker1,∗, Arzu Yildirim2, Arzu Turker2, Meral Yucel1,3

Middle East Technical University, Molecular Biology-Biotechnology&D Center, 06800 Ankara, TurkeyAbant Izzet Baysal University, Department of Biology, 14280 Bolu,urkeyMiddle East Technical University, Department of Biology, 06800

nkara, Turkey

he most of the bioactive compounds are found in trace amountso, an efficient extraction procedure from a given tissue is a

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http://dx.doi.org/10.1016/j.nbt.2012.08.471

ew Biotechnology · Volume 29S · September 2012

ig problem. An efficient quantitative detection coupled to thextraction procedure is necessary in order to cover the whole phe-omena. The increasing dissolution volume of the lyophilizedample may result in dilution of the total content of the pheno-ics. In choosing the analytical measurement method, the lowerample-enjection volume, the seperate detection of the phenolicsaving same UV absorbance, short run time and the sensitivityre the important parameters to prefer LCMSMS compared to thelassical HPLC.

We designed an analytical measurement method via liquidhromatography tandem mass spectrometry with both positivend negative electrospary ionization. MS grade methanol (B) andmM ammonium formate (negative ionisation) with 0.5% formiccid (positive ionisation) (A) were used as mobile phase in gradientrade profile. 5 �L enjection volume was enough for the repetetiveesults. At least 25 different phenolic compounds could be quanti-ed through 13 min run time in 0.01–25 ppm range. The methodas applied for the woodland strawberry samples extracted in

wo different methods together with their upper ground parts.ince there was no a study informing the phenolic content of theoodland strawberry via LCMSMS, it was seen that the optimizedethod is a better alternative compared to HPLC. The method can

e used for any liquid sample for the phenolic profile analyses.Keywords: Phenolics; LCMSMS; Woodland strawberry; HPLC

ttp://dx.doi.org/10.1016/j.nbt.2012.08.469

oster 5.0.30

creening of lipopolysaccharide binding DNA aptamernd its application to fabricating electrochemicalptasensor

ung-Eun Kim∗, Dong-Kwan Kim, Ah-Rum Han, Woo-Seok Choe

School of Chemical Engineering, Sungkyunkwan University, 300hunchun-dong, Suwon 440-746, Republic of Korea

ipopolysaccharide (LPS) is a major component of the outer mem-rane of Gram-negative bacteria and induces a strong immuneesponse leading to a fatal septic shock upon its internalizationnto mammalian cells. Due to its toxicity, fast and accurate detec-ion of even small amount of LPS often entrained with therapeuticnd/or blood products is therefore of primary importance. Despitehe presence of several LPS affinity binders, very few have beenxploited in developing electrochemical sensors capable of selec-ively detecting LPS from crude biological liquors. Using CE-basedon-SELEX (capillary electrophoresis based non-systematic evo-

ution of ligands by exponential enrichment), we identified 10ifferent single stranded DNA aptamers (designated as B1 to B10)howing specific affinity to LPS with dissociation constants (Kd) inhe nanomolar range. Based on the sequence and secondary struc-ure analysis of the LPS binding aptamers, an aptamer exhibitinghe highest affinity to LPS (i.e. B2) was selected as a sensing probe toonstruct an impedance biosensor on a gold electrode. The devel-ped electrochemical aptasensor showed excellent sensitivity andpecificity in the linear detection range from 0.01 to 1 ng/mL of

PS despite the presence of various interfering contaminants suchs plasmid DNA, RNA, BSA, sugars and cholesterol. In comparisonith the traditional Limulus Amoebocyte Lysate (LAL) assay, the

ptasensor required significantly reduced LPS detection time with-ut compromising the dynamic detection range, raising a goodotential for its implementation to crude biological liquors whererapid and specific detection of LPS is of primary concern.

Keywords: Aptamer; Lipopolysaccharide; CE-based non-ELEX; Electrochemical aptasensor

ttp://dx.doi.org/10.1016/j.nbt.2012.08.470

oster 5.0.31

uantitative analysis of different preparations ofuman thyrotropin (hTSH): a comparison between RP-PLC and the in vivo bioassay based on thyroxine

timulation in mice

.E. Almeida, R. Damiani, J.E. Oliveira, M.T.C.P. Ribela, P.artolini∗

Biotechnology Department, IPEN-CNEN, Av. Prof. Lineu Prestes 2242,idade Universitária, 05508-900 São Paulo, Brazil

ith the intention of setting up physico-chemical methods thatan reduce animal use, the hTSH content of different prepa-ations of native, pituitary and of recombinant hormone wasetermined by reversed-phase high performance liquid chro-atography (RP-HPLC) and compared with the data obtained by

he classical mouse in vivo bioassay. Recombinant hTSH producedy Genzyme (Thyrogen®) was used as a reference preparationn both assays. A linear relation was obtained between the two

ethods, with a significant coefficient of correlation: r = 0.8359;< 0.001; n = 16. The corresponding equation was: BA�g = 0.9177P-HPLC�g + 0.4097. Considering, though, that this correlationhould be improved and that a specific individual sample coulde eventually classified as an outlier, we decided to improve theeliability of protein determination based on the BCA assay. Were thus in the process of setting up an in-house calibrator ofTSH (possibly a WHO International Standard), whose molarontent is strictly determined by amino acid composition anal-sis, and of re-determining all protein content of each samplenalyzed. The pharmacokinetics properties and N-glycan struc-ures of a typical pituitary and recombinant hTSH preparationave also been determined and will be studied in the light of

www.elsevier.com/locate/nbt S169