Differential CTX-M Expression from a Conserved Promoter: Role of Promoter-Associated Spacer Sequences Downstream of the blaCTX-M Regulon

Aims: The aim of this project was to explore the different CTX-M expression levels occurring from a single conserved promoter with different spacer sequences, the variation of which is hypothesized to be a key factor in fluctuating levels of CTX-M. Methods: The blaCTX-M promoter fragments with five different spacer sequences were amplified, sequenced and cloned into the pUA66 expression vector carrying the green fluorescent protein (GFP) gene. The expression of blaCTX-M in the transconjugants was analyzed using fluorescence microscopy, flow cytometry and qRT-PCR. Results: The promoters of all the blaCTX-M genes were provided by ISEcp1 and were extremely conserved. The promoter-associated spacer sequences varied from 42 to 127 bp and variations in GFP expression in the five transconjugants were observed. A nucleic acid deletion and point mutation were detected in the spacer sequences by variations in which the expression of blaCTX-M was influenced. Conclusion: The different spacer sequences have a significant impact on the activity of the conserved promoter. The shorter spacer sequence between the conserved promoter and the blaCTX-M gene does not specifically enhance the expression of blaCTX-M, contrary to previous reports. The expression of blaCTX-M may be regulated by changes in promoter activity caused by diverse spacer sequences.

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Transfer of Green Fluorescent Protein (GFP) Gene to Betta splendens Embryos by Transfection and Electroporation Methods

Omni-Akuatika ◽

10.20884/1.oa.2018.14.2.556 ◽

2018 ◽

Vol 14 (2) ◽

Author(s):

Eni Kusrini ◽

Alimuddin Alimuddin ◽

Erma Primanita Hayuningtyas ◽

Syuhada Restu Danupratama

Keyword(s):

Green Fluorescent Protein ◽

Gene Transfer ◽

Expression Vector ◽

Fluorescent Protein ◽

Betta Splendens ◽

Foreign Gene ◽

Gfp Expression ◽

Dna Concentration ◽

Gfp Gene ◽

Green Fluorescent

Transfection and electroporation method shave a high possibility to apply towards transgenic production of small eggs size fish species. This study aimed to examine the potential of transfection and electroporation methods to use for transferring a foreign gene into betta fish (Betta splendens) embryos using green fluorescent protein (GFP) gene as a model. Fish were spawned naturally in the ratio of male: female was 1:1, then a total of 200 eggs were taken for each treatment. Transfection was performed for 30 minutes (room temperature of about 25 °C) at two-cell stage of embryos using transfast reagent. Transfection reaction consisted of 0.75 µL transfast reagent, 0.25 µL GFP expression vector (DNA concentration: 50 µg/µL) and 99 µL NaCl solution (concentration: 0,95%). Electroporation was performed using 125 volt cm-1, 3 times pulse frequency at one second interval and pulse length of 7 micro seconds. A volume of 800 µL GFP expression vector solution (DNA concentration: 50 µg/ µL) in PBS was used for electroporation. The successful of foreign gene transfer was determined by PCR method with GFP specific primers. The results showed that hatching rate of eggs in transfection treatment was 67.08%, while the electroporation was 72.09%. Survival of larvae in transfection treatment was 73.00%, while the electroporation was 75.00%. The results of PCR analysis showed that transfection method allowed 65% of the survived fish carrying GFP gene, whereas the electroporation method was 70%. Thus, foreign gene transfer in betta fish can be conducted using the transfection and electroporation methods.

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Expanding the Genetic Toolbox for Leptospira Species by Generation of Fluorescent Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.02199-10 ◽

2010 ◽

Vol 76 (24) ◽

pp. 8135-8142 ◽

Cited By ~ 14

Author(s):

Florence Aviat ◽

Leyla Slamti ◽

Gustavo M. Cerqueira ◽

Kristel Lourdault ◽

Mathieu Picardeau

Keyword(s):

Molecular Basis ◽

Promoter Activity ◽

Expression Vector ◽

Fluorescent Protein ◽

Bacterial Species ◽

Gfp Expression ◽

Microplate Reader ◽

Green Fluorescent ◽

Leptospira Species

ABSTRACT Our knowledge of the genetics and molecular basis of the pathogenesis associated with Leptospira, in comparison to those of other bacterial species, is very limited. An improved understanding of pathogenic mechanisms requires reliable genetic tools for functional genetic analysis. Here, we report the expression of gfp and mRFP1 genes under the control of constitutive spirochetal promoters in both saprophytic and pathogenic Leptospira strains. We were able to reliably measure the fluorescence of Leptospira by fluorescence microscopy and a fluorometric microplate reader-based assay. We showed that the expression of the gfp gene had no significant effects on growth in vivo and pathogenicity in L. interrogans. We constructed an expression vector for L. biflexa that contains the lacI repressor, an inducible lac promoter, and gfp as the reporter, demonstrating that the lac system is functional in Leptospira. Green fluorescent protein (GFP) expression was induced by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) in L. biflexa transformants harboring the expression vector. Finally, we showed that GFP can be used as a reporter to assess promoter activity in different environmental conditions. These results may facilitate further advances for studying the genetics of Leptospira spp.

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Role of Untranslated Regions in Regulation of Gene Expression, Replication, and Pathogenicity of Newcastle Disease Virus Expressing Green Fluorescent Protein

Journal of Virology ◽

10.1128/jvi.02049-09 ◽

2009 ◽

Vol 84 (5) ◽

pp. 2629-2634 ◽

Cited By ~ 16

Author(s):

Shin-Hee Kim ◽

Siba K. Samal

Keyword(s):

Gene Expression ◽

Green Fluorescent Protein ◽

Newcastle Disease Virus ◽

Newcastle Disease ◽

Fluorescent Protein ◽

Untranslated Regions ◽

Foreign Gene ◽

Gfp Expression ◽

Green Fluorescent

ABSTRACT To gain insight into the role of untranslated regions (UTRs) in regulation of foreign gene expression, replication, and pathogenicity of Newcastle disease virus (NDV), a green fluorescent protein (GFP) gene flanked by 5′ and 3′ UTRs of each NDV gene was individually expressed by recombinant NDVs. UTRs of each gene modulated GFP expression positively or negatively. In particular, UTRs of the M and F genes enhanced levels of GFP expression at the junction of the P and M genes without altering replication of NDV, suggesting that UTRs could be used for enhanced expression of a foreign gene by NDV.

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