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Opimization of the condition for purificatoin of non-specific lipid transfer protein2 (nsLTP2) from transformed E. coli (BL21 strain)
Abstracts
Poster – [A-10-319-2] Construction disruption cassettes for gene silencing Fateme Nafian Dehkordi, Somaye Mazaheri Alzahra University, Tehran, Iran E-mail addresses: [email protected] (F.N. Dehkordi), [email protected] (S. Mazaheri) Introduction: Disruption cassettes are one of the ways to silencing of gene as targeted in metabolic engineering. Accordingly, it is used for the homologous recombination between the flank sequences of specific marker and targeted gene and of course must be proportional between the length of these regions and homologous recombination frequency in organism. Method: By design primers and PCR, we made Cre-Loxp systems for multiple gene destructors in high-ploid and prototrophic nature of industrial strains so that Loxp sequences are to be around of marker, while homologous region is in flank of them and Cre gene is also added to plasmid. Other method is based on 5′UTR-3′UTR of target gene. So that, 5′UTR directed repeats to be around of marker, 3′tag amplified of 3′UTR is added at the 3′-marker. Results: These methods facilitate the marker rescue and reuse and thus offer efficient way to create multiple gene deletions in a short period of time. Moreover, the use of a heterologous selectable marker makes it possible to disrupt a specific gene in wild type or industrial strains devoid of any auxothrophic marker. The effects caused by the loss of gene functions on physiological processes are studied. Conclusion: If prevention of chromosome rearrangement and incomplete separation of the chromatids during metaphase are done, this system is useful in the introduction of targeted gene deletions in industrial strains. Keywords: Disruption cassettes, Cre-Loxp systems, PCR, Marker
S279
Keywords: PPARγ-1, Spinal cord injury, Primer, Real-time PCR doi:10.1016/j.clinbiochem.2011.08.691
Poster – [A-10-327-1] Opimization of the condition for purificatoin of non-specific lipid transfer protein2 (nsLTP2) from transformed E. coli (BL21 strain) Nahid Zainodini, Mehran Miroliaei, Kamran Ghaedi, Jamal Moshtaghian Seyed Division of Molecular Cell Biology, Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran E-mail address: [email protected] (N. Zainodini) Introduction: Non-specific lipid transfer proteins (nsLTP) are small basic and secreted proteins. Based on the molecular weight they are divided into two families: nsLTP1 and nsLTP2. They have ability to facilitate transfer of lipids between membranes in vitro. During this project CDS sequence for nsLTP from rice was cloned into pGEX-6p-2 plasmid containing the lac promoter, which was then transformed into the bacterium Escherichia coli (BL21 strain). Isopropyl β-d-1-thiogalactopyranoside (IPTG) induction experiment was performed using various concentrations of IPTG and incubation times for efficient increases in expression of LTP. Conclusions: Data indicated the optimized concentration for IPTG was in average of 0.5–1 mM which could be done for efficient purification of this protein. Keywords: Lipid transfer protein, nsLTP, IPTG, Protein expression, Isopropyl β-d-1-thiogalactopyranoside doi:10.1016/j.clinbiochem.2011.08.692
doi:10.1016/j.clinbiochem.2011.08.690
Poster – [A-10-322-1] Optimization of real-time PCR conditions to assess PPARγ expression level in spinal cord injuries Esmat Mohammadi, Kamran Ghaedi, Abolghasem Esmaeili, Soheila Rahgozar Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran E-mail address: [email protected] (E. Mohammadi) Introduction: Traumatic spinal cord injury is a complicated process which results in occasional bleeding and cell death at the site of stroke. This phenomenon causes activation of secondary pathways which leads to inflammation, and neurodegeneration. Since PPARγ agonists have been recently used for decrease of neuronal inflammation and increase in neuronal survival rates. The aim of this study is chasing the changes in expression of this transcription factor after different stages of spinal cord injuries. Methods: As real time PCR technique is a powerful tool for analysis of expression level, we designed specific primers for isoform 1 using oligo 6 and Beacon designer softwares. To optimize the conditions of experiments a control rat was selected and the thoracic segment-9 was isolated for RNA extraction, cDNA synthesis was carried out and followed by RT-PCR. Results and conclusion: Data indicated in control samples, the expression of PPARγ is not significantly related to the house keeping gene (β-tubulin), thus real time PCR data indicated that the threshold cycle for PPARγ is 30.43 while for tubulin is 18.96. Thus we are going to do further experiment based on the above condition.
E.poster – [A-10-342-1] Construction of a minicircle DNA vector carrying enhanced green fluorescence protein (egfp) gene Nafise Sanei, Kamran Ghaedi, Yahya Khazaie, Kyanoosh Dormiani, Mahboube Forouzanfar, Manoochehr Tavassoli, Hossein Nasr Esfahani Mohammad Isfahan, Salman Farsi Avenue, Mehr Street, Alikhani Alley, Isfahan Royan Institute, Isfahan, Iran E-mail addresses: [email protected] (N. Sanei), [email protected] (K. Ghaedi), [email protected] (Y. Khazaie), [email protected] (K. Dormiani), [email protected] (M. Forouzanfar), [email protected] (M. Tavassoli), [email protected] (H.N.E. Mohammad) Introduction: The loss of transgene expression has been a major obstacle in the use of nonviral vectors in vivo because the bacterial DNA linked to a mammalian expression cassette resulted in transcriptional silencing of the transgene in vivo by formation of repressive heterochromatin on the plasmid DNA backbone, which then spreads and inactivates the transgene. In the minicircle vectors with a phage ΦC31 integrase-mediated intramolecular recombination the bacterial backbone is removed. Minicircle vectors are capable of expressing high and persistent levels of genes in vivo. In this study we constructed a minicircle vector carrying egfp. Material and method: First, we amplified egfp from the plasmid pEGFP-C1 and inserted the PCR product into SnaI site of pBAD.gIIIA. The attp and attb sequences were added into the egfp primers. The 5.5 kb band presents recombinant plasmid pBAD.EGFP. Then we
Poster – [A-10-319-2] Construction disruption cassettes for gene silencing Fateme Nafian Dehkordi, Somaye Mazaheri Alzahra University, Tehran, Iran E-mail addresses: [email protected] (F.N. Dehkordi), [email protected] (S. Mazaheri) Introduction: Disruption cassettes are one of the ways to silencing of gene as targeted in metabolic engineering. Accordingly, it is used for the homologous recombination between the flank sequences of specific marker and targeted gene and of course must be proportional between the length of these regions and homologous recombination frequency in organism. Method: By design primers and PCR, we made Cre-Loxp systems for multiple gene destructors in high-ploid and prototrophic nature of industrial strains so that Loxp sequences are to be around of marker, while homologous region is in flank of them and Cre gene is also added to plasmid. Other method is based on 5′UTR-3′UTR of target gene. So that, 5′UTR directed repeats to be around of marker, 3′tag amplified of 3′UTR is added at the 3′-marker. Results: These methods facilitate the marker rescue and reuse and thus offer efficient way to create multiple gene deletions in a short period of time. Moreover, the use of a heterologous selectable marker makes it possible to disrupt a specific gene in wild type or industrial strains devoid of any auxothrophic marker. The effects caused by the loss of gene functions on physiological processes are studied. Conclusion: If prevention of chromosome rearrangement and incomplete separation of the chromatids during metaphase are done, this system is useful in the introduction of targeted gene deletions in industrial strains. Keywords: Disruption cassettes, Cre-Loxp systems, PCR, Marker
S279
Keywords: PPARγ-1, Spinal cord injury, Primer, Real-time PCR doi:10.1016/j.clinbiochem.2011.08.691
Poster – [A-10-327-1] Opimization of the condition for purificatoin of non-specific lipid transfer protein2 (nsLTP2) from transformed E. coli (BL21 strain) Nahid Zainodini, Mehran Miroliaei, Kamran Ghaedi, Jamal Moshtaghian Seyed Division of Molecular Cell Biology, Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran E-mail address: [email protected] (N. Zainodini) Introduction: Non-specific lipid transfer proteins (nsLTP) are small basic and secreted proteins. Based on the molecular weight they are divided into two families: nsLTP1 and nsLTP2. They have ability to facilitate transfer of lipids between membranes in vitro. During this project CDS sequence for nsLTP from rice was cloned into pGEX-6p-2 plasmid containing the lac promoter, which was then transformed into the bacterium Escherichia coli (BL21 strain). Isopropyl β-d-1-thiogalactopyranoside (IPTG) induction experiment was performed using various concentrations of IPTG and incubation times for efficient increases in expression of LTP. Conclusions: Data indicated the optimized concentration for IPTG was in average of 0.5–1 mM which could be done for efficient purification of this protein. Keywords: Lipid transfer protein, nsLTP, IPTG, Protein expression, Isopropyl β-d-1-thiogalactopyranoside doi:10.1016/j.clinbiochem.2011.08.692
doi:10.1016/j.clinbiochem.2011.08.690
Poster – [A-10-322-1] Optimization of real-time PCR conditions to assess PPARγ expression level in spinal cord injuries Esmat Mohammadi, Kamran Ghaedi, Abolghasem Esmaeili, Soheila Rahgozar Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran E-mail address: [email protected] (E. Mohammadi) Introduction: Traumatic spinal cord injury is a complicated process which results in occasional bleeding and cell death at the site of stroke. This phenomenon causes activation of secondary pathways which leads to inflammation, and neurodegeneration. Since PPARγ agonists have been recently used for decrease of neuronal inflammation and increase in neuronal survival rates. The aim of this study is chasing the changes in expression of this transcription factor after different stages of spinal cord injuries. Methods: As real time PCR technique is a powerful tool for analysis of expression level, we designed specific primers for isoform 1 using oligo 6 and Beacon designer softwares. To optimize the conditions of experiments a control rat was selected and the thoracic segment-9 was isolated for RNA extraction, cDNA synthesis was carried out and followed by RT-PCR. Results and conclusion: Data indicated in control samples, the expression of PPARγ is not significantly related to the house keeping gene (β-tubulin), thus real time PCR data indicated that the threshold cycle for PPARγ is 30.43 while for tubulin is 18.96. Thus we are going to do further experiment based on the above condition.
E.poster – [A-10-342-1] Construction of a minicircle DNA vector carrying enhanced green fluorescence protein (egfp) gene Nafise Sanei, Kamran Ghaedi, Yahya Khazaie, Kyanoosh Dormiani, Mahboube Forouzanfar, Manoochehr Tavassoli, Hossein Nasr Esfahani Mohammad Isfahan, Salman Farsi Avenue, Mehr Street, Alikhani Alley, Isfahan Royan Institute, Isfahan, Iran E-mail addresses: [email protected] (N. Sanei), [email protected] (K. Ghaedi), [email protected] (Y. Khazaie), [email protected] (K. Dormiani), [email protected] (M. Forouzanfar), [email protected] (M. Tavassoli), [email protected] (H.N.E. Mohammad) Introduction: The loss of transgene expression has been a major obstacle in the use of nonviral vectors in vivo because the bacterial DNA linked to a mammalian expression cassette resulted in transcriptional silencing of the transgene in vivo by formation of repressive heterochromatin on the plasmid DNA backbone, which then spreads and inactivates the transgene. In the minicircle vectors with a phage ΦC31 integrase-mediated intramolecular recombination the bacterial backbone is removed. Minicircle vectors are capable of expressing high and persistent levels of genes in vivo. In this study we constructed a minicircle vector carrying egfp. Material and method: First, we amplified egfp from the plasmid pEGFP-C1 and inserted the PCR product into SnaI site of pBAD.gIIIA. The attp and attb sequences were added into the egfp primers. The 5.5 kb band presents recombinant plasmid pBAD.EGFP. Then we