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Construction of specifically expressed vector in mammary gland for lacS and its transfection into bovine fetal fibroblasts mediated by liposome
New Biotechnology · Volume 29S · September 2012
term goal is the identification of microorganisms which either themselves or whose enzymes could be used as feed additives for gastrointestinal fumonisin detoxification. Previously, we isolated the fumonisin degrading alphaproteobacterium Sphingopyxis sp. MTA144. Our present goal was the characterization of this strain regarding its fumonisin catabolism. We found that the hydrolysis of the mycotoxin in the clear lysate of the strain was much faster when the biomass was induced with 10 mg/l FB1 prior to the decomposition experiment itself. We concluded that the fum genes are not constitutively expressed, but inducible. Two genes for transcriptional regulators in the fum gene cluster may be responsible for the observed gene expression regulation. FB1 degradation experiments with biomass of strain MTA144 showed that FB1 was not only catabolised in mineral medium, but also in complex growth medium, indicating that there is little or no catabolite repression. Moreover, we found that the presence of up to 100 mg/l FB1 in the medium promoted growth of Sphingopyxis sp. MTA144, but had no effect on related strains which do not catabolise fumonisins. This indicates that Sphingopyxis sp. MTA144 degrades FB1 for energy gain or as a carbon source rather than for self-protecting detoxification. http://dx.doi.org/10.1016/j.nbt.2012.08.466 Poster 5.0.27 Construction of specifically expressed vector in mammary gland for lacS and its transfection into bovine fetal fibroblasts mediated by liposome Huanmin Zhou∗ , Lu Li College of Life Science, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, PR China This study was designed to optimize conditions for transfecting a mammary gland specific transgene into bovine fetal fibroblasts. A 0.92 kb fragment of bovine -lactoglobulin gene sequence was obtained from bovine genome by PCR amplification and inserted into the T site of pMD19-Simple T plasmid. A 1.49 kb of lacS gene coding sequence was cloned from sulfolobus solfataricus genome by PCR amplification and inserted into the pUC19 plasmid. The coding sequence of Neor were derived by PCR amplification from pIRES2-EGFP plasmid and inserted into the BamHI/NheI site of pIRES2-EGFP plasmid. The resultant vector (pNIE) contained a Neor and an EGFP gene, which were linked by an internal ribosome entry site (IRES) sequence downstream of the cytomegalovirus (CMV) promoter. Finally, the vector pNIE was assembled into the pUC19 plasmid thus creating a pBLI vector, which contained the Neor and EGFP gene regulated by CMV promoter for expression in a non-tissue specific mode, and the lacS gene regulated by bovine -lactoglobulin promoter for specific expression in mammary gland. 2 × 105 bovine fetal fibroblasts (bFF) cells were transfected in DMEM with pBLI using TRANSfection for 48 h. The transfected cells were cultured for 48 h before adding G418 at concentrations of 800 g/mL for 14 d. The results shown that 1.0 g pBLI plasmid and 3 L TRANSfection yielded the desirable efficiency of transfection, which indicated that a specifically expressed-vector in mammary gland for lacS gene was successfully constructed,
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transfection parameters were developed and an efficient screening measures were established for detecting transgenic somatic cells. http://dx.doi.org/10.1016/j.nbt.2012.08.467 Poster 5.0.28 Structural and functional analysis of lipase gene of Amsacta moorei entomopoxvirus Emine Ozsahin∗ , Kazim Sezen, Zihni Demirbag Karadeniz Technical University, Faculty of Science, Department of Biology, Trabzon, Turkey The entomopoxvirus isolated from the larvae of the moth, Amsacta moorei, is a distant relative of the better studied orthopoxviruses which include variola and vaccinia virus. A. moorei entomopoxvirus (AmEPV) has 279 unique open reading frames (ORF). Among these, AMV133 has 864 nt coding for a protein of 288 amino acids. Sequence derived amino acid analysis of AMV133 suggested it to be a putative triacylglyceride lipase gene, which could conceivably function as a virulence gene through lipid hydrolysis. AMV133 was transcribed as delayed early class of temporal cascade since it was not inhibited by an inhibitor of DNA synthesis, however it effected by a protein synthesis inhibitor. Transcription started 6th hours post infection and continued until 72 hpi. 5 -RACE analysis showed that transcription initiated at position –77, relative to the translational start site of this gene. To determine the limits of the putative promoters, upstream sequences of various lengths were cloned in front of a firefly luciferase reporter gene. The resulting plasmid constructs were tested in a dual assay. The promoter activity was lost when the length of the sequence upstream of the translational start site was reduced from −82 to −21 nucleotides. Lipase gene of AmEPV was cloned and expressed in both baculovirus and bacterial expression vector systems. Purified protein was used for lipase assay. The results show that the purified protein has lipase activity. Keywords: Amsacta moorei entomopoxvirus; Lipase; Transcriptomic; Promoter analysis http://dx.doi.org/10.1016/j.nbt.2012.08.468 Poster 5.0.29 An LCMSMS method to analyse phenolic profile in the liquid extract, with woodland strawberry (Fragaria vesca l.) application Tamay S¸eker1,∗ , Arzu Yildirim2 , Arzu Turker2 , Meral Yucel1,3 1 Middle East Technical University, Molecular Biology-Biotechnology R&D Center, 06800 Ankara, Turkey 2 Abant I˙zzet Baysal University, Department of Biology, 14280 Bolu, Turkey 3 Middle East Technical University, Department of Biology, 06800 Ankara, Turkey
The most of the bioactive compounds are found in trace amounts so, an efficient extraction procedure from a given tissue is a
term goal is the identification of microorganisms which either themselves or whose enzymes could be used as feed additives for gastrointestinal fumonisin detoxification. Previously, we isolated the fumonisin degrading alphaproteobacterium Sphingopyxis sp. MTA144. Our present goal was the characterization of this strain regarding its fumonisin catabolism. We found that the hydrolysis of the mycotoxin in the clear lysate of the strain was much faster when the biomass was induced with 10 mg/l FB1 prior to the decomposition experiment itself. We concluded that the fum genes are not constitutively expressed, but inducible. Two genes for transcriptional regulators in the fum gene cluster may be responsible for the observed gene expression regulation. FB1 degradation experiments with biomass of strain MTA144 showed that FB1 was not only catabolised in mineral medium, but also in complex growth medium, indicating that there is little or no catabolite repression. Moreover, we found that the presence of up to 100 mg/l FB1 in the medium promoted growth of Sphingopyxis sp. MTA144, but had no effect on related strains which do not catabolise fumonisins. This indicates that Sphingopyxis sp. MTA144 degrades FB1 for energy gain or as a carbon source rather than for self-protecting detoxification. http://dx.doi.org/10.1016/j.nbt.2012.08.466 Poster 5.0.27 Construction of specifically expressed vector in mammary gland for lacS and its transfection into bovine fetal fibroblasts mediated by liposome Huanmin Zhou∗ , Lu Li College of Life Science, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, PR China This study was designed to optimize conditions for transfecting a mammary gland specific transgene into bovine fetal fibroblasts. A 0.92 kb fragment of bovine -lactoglobulin gene sequence was obtained from bovine genome by PCR amplification and inserted into the T site of pMD19-Simple T plasmid. A 1.49 kb of lacS gene coding sequence was cloned from sulfolobus solfataricus genome by PCR amplification and inserted into the pUC19 plasmid. The coding sequence of Neor were derived by PCR amplification from pIRES2-EGFP plasmid and inserted into the BamHI/NheI site of pIRES2-EGFP plasmid. The resultant vector (pNIE) contained a Neor and an EGFP gene, which were linked by an internal ribosome entry site (IRES) sequence downstream of the cytomegalovirus (CMV) promoter. Finally, the vector pNIE was assembled into the pUC19 plasmid thus creating a pBLI vector, which contained the Neor and EGFP gene regulated by CMV promoter for expression in a non-tissue specific mode, and the lacS gene regulated by bovine -lactoglobulin promoter for specific expression in mammary gland. 2 × 105 bovine fetal fibroblasts (bFF) cells were transfected in DMEM with pBLI using TRANSfection for 48 h. The transfected cells were cultured for 48 h before adding G418 at concentrations of 800 g/mL for 14 d. The results shown that 1.0 g pBLI plasmid and 3 L TRANSfection yielded the desirable efficiency of transfection, which indicated that a specifically expressed-vector in mammary gland for lacS gene was successfully constructed,
S168
www.elsevier.com/locate/nbt
transfection parameters were developed and an efficient screening measures were established for detecting transgenic somatic cells. http://dx.doi.org/10.1016/j.nbt.2012.08.467 Poster 5.0.28 Structural and functional analysis of lipase gene of Amsacta moorei entomopoxvirus Emine Ozsahin∗ , Kazim Sezen, Zihni Demirbag Karadeniz Technical University, Faculty of Science, Department of Biology, Trabzon, Turkey The entomopoxvirus isolated from the larvae of the moth, Amsacta moorei, is a distant relative of the better studied orthopoxviruses which include variola and vaccinia virus. A. moorei entomopoxvirus (AmEPV) has 279 unique open reading frames (ORF). Among these, AMV133 has 864 nt coding for a protein of 288 amino acids. Sequence derived amino acid analysis of AMV133 suggested it to be a putative triacylglyceride lipase gene, which could conceivably function as a virulence gene through lipid hydrolysis. AMV133 was transcribed as delayed early class of temporal cascade since it was not inhibited by an inhibitor of DNA synthesis, however it effected by a protein synthesis inhibitor. Transcription started 6th hours post infection and continued until 72 hpi. 5 -RACE analysis showed that transcription initiated at position –77, relative to the translational start site of this gene. To determine the limits of the putative promoters, upstream sequences of various lengths were cloned in front of a firefly luciferase reporter gene. The resulting plasmid constructs were tested in a dual assay. The promoter activity was lost when the length of the sequence upstream of the translational start site was reduced from −82 to −21 nucleotides. Lipase gene of AmEPV was cloned and expressed in both baculovirus and bacterial expression vector systems. Purified protein was used for lipase assay. The results show that the purified protein has lipase activity. Keywords: Amsacta moorei entomopoxvirus; Lipase; Transcriptomic; Promoter analysis http://dx.doi.org/10.1016/j.nbt.2012.08.468 Poster 5.0.29 An LCMSMS method to analyse phenolic profile in the liquid extract, with woodland strawberry (Fragaria vesca l.) application Tamay S¸eker1,∗ , Arzu Yildirim2 , Arzu Turker2 , Meral Yucel1,3 1 Middle East Technical University, Molecular Biology-Biotechnology R&D Center, 06800 Ankara, Turkey 2 Abant I˙zzet Baysal University, Department of Biology, 14280 Bolu, Turkey 3 Middle East Technical University, Department of Biology, 06800 Ankara, Turkey
The most of the bioactive compounds are found in trace amounts so, an efficient extraction procedure from a given tissue is a