Fitness cost of the green fluorescent protein in gastrointestinal bacteria

2003 ◽  
Vol 49 (9) ◽  
pp. 531-537 ◽  
Author(s):  
Camilla Rang ◽  
James E Galen ◽  
James B Kaper ◽  
Lin Chao
Keyword(s):  
Green Fluorescent Protein ◽  
Doubling Time ◽  
Fluorescent Protein ◽  
Shigella Flexneri ◽  
Bacterial Species ◽  
Enteric Bacteria ◽  
Fitness Cost ◽  
Gfp Expression ◽  
Selection Experiments ◽  
Green Fluorescent

There are surprisingly few studies that have successfully used the green fluorescent protein (GFP) as a quantitative reporter in selection experiments screening for inducible bacterial promoters. One explanation is that GFP expression may confer a fitness cost for bacteria. To test this possibility, we monitored the doubling time in enteric bacteria expressing GFP. Four bacterial species, Escherichia coli, enterohaemorrhagic E. coli, Shigella flexneri, Salmonella typhi, and Vibrio cholerae, were examined. The level of GFP expression was varied by using a salt-inducible promoter. After accounting for the increase in doubling time resulting from elevated osmolarity, the doubling time of all bacteria was found to increase proportionally with GFP expression, and some strains were more affected than others. Cultures of the bacteria most affected by GFP exhibited a proportion of elongated cells, which suggests that GFP production could interfere with cell division in these strains. The results in this study show that GFP is costly to bacteria and suggest that overly active promoters should be difficult to obtain from a genomic promoter library. They also suggest that the chances of succeeding in using GFP as a reporter in selection experiments are increased by growing the bacteria for the fewest number of generations and by subduing the expression of GFP whenever possible, such as by using a low copy vector to clone the library.Key words: green fluorescent protein, protein cost, promoter activity.

Green fluorescent protein (GFP) expression patterns in the olfactory epithelium of GFP transgenic cloned Jinhua pigs

2013 ◽  
Vol 95 (3) ◽  
pp. 319-329
Author(s):  
Atsushi Hirao ◽  
Tatsuo Kawarasaki ◽  
Kenjiro Konno ◽  
Satoko Enya ◽  
Masatoshi Shibata ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Olfactory Epithelium ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Gfp Expression ◽  
Green Fluorescent

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

Omni-Akuatika ◽  
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. 

scholarly journals Construction of Self-Transmissible Green Fluorescent Protein-Based Biosensor Plasmids and Their Use for Identification of N-Acyl Homoserine-Producing Bacteria in Lake Sediments

2010 ◽  
Vol 76 (18) ◽  
pp. 6119-6127 ◽  
Author(s):  
Putthapoom Lumjiaktase ◽  
Claudio Aguilar ◽  
Tom Battin ◽  
Kathrin Riedel ◽  
Leo Eberl
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Natural Habitat ◽  
Homoserine Lactone ◽  
Signal Molecules ◽  
Broad Host Range ◽  
Construction Of Self ◽  
Green Fluorescent ◽  
The Impact

ABSTRACT Many bacteria utilize quorum sensing (QS) systems to communicate with each other by means of the production, release, and response to signal molecules. N-Acyl homoserine lactone (AHL)-based QS systems are particularly widespread among the Proteobacteria, in which they regulate various functions. It has become evident that AHLs can also serve as signals for interspecies communication. However, knowledge on the impact of AHLs for the ecology of bacteria in their natural habitat is scarce, due mainly to the lack of tools that allow the study of QS in bacterial communities in situ. Here, we describe the construction of self-mobilizable green fluorescent protein (GFP)-based AHL sensors that utilize the conjugation and replication properties of the broad-host-range plasmid RP4. We show that these novel AHL sensor plasmids can be easily transferred to different bacterial species by biparental mating and that they give rise to green fluorescent cells in case the recipient is an AHL producer. We also demonstrate that these sensor plasmids are capable of self-spreading within mixed biofilms and are a suitable tool for the identification of AHL-producing bacteria in lake sediment.

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

2016 ◽  
Vol 26 (4) ◽  
pp. 284-290 ◽  
Author(s):  
Lin Liu ◽  
Xiangyan Zhang ◽  
Siyuan Yang ◽  
Yao Zhai ◽  
Weijia Liu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Promoter Activity ◽  
Expression Vector ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Qrt Pcr ◽  
Key Factor ◽  
Green Fluorescent

<b><i>Aims:</i></b> 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. <b><i>Methods:</i></b> The <i>bla</i><sub>CTX-M</sub> 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 <i>bla</i><sub>CTX-M</sub> in the transconjugants was analyzed using fluorescence microscopy, flow cytometry and qRT-PCR. <b><i>Results:</i></b> The promoters of all the <i>bla</i><sub>CTX-M</sub> genes were provided by IS<i>Ecp1 </i>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 <i>bla</i><sub>CTX-M</sub> was influenced. <b><i>Conclusion:</i></b> 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 <i>bla</i><sub>CTX-M</sub> gene does not specifically enhance the expression of<i> bla</i><sub>CTX-M</sub>, contrary to previous reports. The expression of <i>bla</i><sub>CTX-M</sub> may be regulated by changes in promoter activity caused by diverse spacer sequences.

scholarly journals Visualizing Viral Dissemination in the Mouse Nervous System, Using a Green Fluorescent Protein-Expressing Borna Disease Virus Vector

2010 ◽  
Vol 84 (10) ◽  
pp. 5438-5442 ◽  
Author(s):  
Andreas Ackermann ◽  
Timo Guelzow ◽  
Peter Staeheli ◽  
Urs Schneider ◽  
Bernd Heimrich
Keyword(s):  
Nervous System ◽  
Green Fluorescent Protein ◽  
Disease Virus ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Borna Disease Virus ◽  
Viral Dissemination ◽  
Viral Spread ◽  
Green Fluorescent ◽  
Borna Disease

ABSTRACT Borna disease virus (BDV) frequently persists in the brain of infected animals. To analyze viral dissemination in the mouse nervous system, we generated a mouse-adapted virus that expresses green fluorescent protein (GFP). This viral vector supported GFP expression for up to 150 days and possessed an extraordinary staining capacity, visualizing complete dendritic arbors as well as individual axonal fibers of infected neurons. GFP-positive cells were first detected in cortical areas from where the virus disseminated through the entire central nervous system (CNS). Late in infection, GFP expression was found in the sciatic nerve, demonstrating viral spread from the central to the peripheral nervous system.

63 ESTABLISHMENT OF GREEN FLUORESCENT PROTEIN EXPRESSED DOG CELL LINES CONTROLLED BY DOXYCYCLINE

2010 ◽  
Vol 22 (1) ◽  
pp. 190
Author(s):  
M. J. Kim ◽  
H. J. Oh ◽  
J. E. Park ◽  
S. G. Hong ◽  
J. E. Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cell Line ◽  
Cell Size ◽  
Fluorescent Protein ◽  
Biomedical Science ◽  
Gfp Expression ◽  
Cell Stage ◽  
Fetal Fibroblasts ◽  
Green Fluorescent

An inducible gene expression system in transgenic animals has been widely used in biomedical science. The aim of this study was to establish green fluorescent protein (GFP) inducible dog cell line and evaluate the system in embryos using interspecies somatic cell nuclear transfer (iSCNT). Canine fetal fibroblasts were transfected with retroviral vector containing GFP, rtTA, and TRE and designated Gteton cell line. For iSCNT, bovine ovaries were collected from a local slaughterhouse and COCs were matured for 24 h. The denuded oocytes were enucleated, injected with Gteton cells, treated with 24 h of doxycycline (DOX), and electrically fused (NEPA GENE, 34 V, 15 μs, 2 pulses). The reconstructed oocytes were activated and then cultured in modified SOF medium. To verify the stability of the Gteton cells, 2 experiments were designed. Experiment 1 was designed to compare the cell size and viability of Gteton and nontransfected cells. Countness™ (Invitrogen, version 1.0, Carlsbad, CA, USA) was used for analysis. In experiment 2, the control of GFP gene expression was observed when the cells were cultured with 1 mg mL-1 of DOX. The cells were also cultured without DOX after 24 h of DOX treatment. Photographs were taken of cultured cells every 12 h. The intensity of GFP expression was analyzed by using Image J freeware (U.S. National Institutes of Health, version 1.42, NIH, Bethesda, MD, USA). To evaluate the reprogramming ability of the Gteton cells in embryos, another 2 experimental designs were planned. Experiment 3 estimated GFP expression in iSCNT embryos when they were cultured with and without DOX. Experiment 4 assessed the development of the iSCNT embryos under microscopy. Data were analyzed using statistical analysis system program (version 9.1, SAS Institute, Cary, NC, USA). In experiment 1, there was no significance (P < 0.05) in average viable cell size (13.7 v. 13.2 μm) or viability (97.0 v. 98.7%). In experiment 2, the GFP intensity increased steadily when cultured in medium containing DOX. The intensity was increased approximately two times after 24 h compared with 12 h of treatment. The intensity after 24 h of DOX treatment decreased to the basal level after 5 days. In experiment 3, the GFP intensity of iSCNT embryos cultured in mSOF containing DOX was increased approximately two times in 16-cell stage compared with 2-cell stage. In experiment 4, the cleavage rate was not significantly different between the 2 groups. In conclusion, we dtermined that the inducible system of Gteton cell line was established in a stable manner. Furthermore the results from iSCNT may indicate the possibility to produce GFP-expressed transgenic puppies controlled by doxycyline. This study was supported by Korean MEST through KOSEF (grant # M10625030005-09N250300510) and BK21 program, RNL BIO, and Natural Balance Korea.

Efficient genetic transformation of Sorghum using a visual screening marker

Genome ◽  
2005 ◽  
Vol 48 (2) ◽  
pp. 321-333 ◽  
Author(s):  
Zhensheng Gao ◽  
J Jayaraj ◽  
S Muthukrishnan ◽  
Larry Claflin ◽  
G H Liang
Keyword(s):  
Green Fluorescent Protein ◽  
Transgenic Plants ◽  
Reporter Gene ◽  
Target Gene ◽  
Fluorescent Protein ◽  
Efficient Production ◽  
Gfp Expression ◽  
Visual Screening ◽  
Green Fluorescent ◽  
Screening Marker

To transform grain sorghum (Sorghum bicolor (L.) Moench) with a visual reporter gene (gfp) and a target gene (tlp), three genotypes (two inbreds, Tx 430 and C401, and a commercial hybrid, Pioneer 8505) were used. We obtained a total of 1011 fertile transgenic plants from 61 independent callus lines, which were produced from 2463 zygotic immature embryos via Agrobacterium-mediated transformation. The reporter gene, gfp, encoding green fluorescent protein (GFP), was used as a visual screening marker, and the target gene, tlp, encoding thaumatin-like protein (TLP), was chosen for enhancing resistance to fungal diseases and drought. Both genes were under the control of the maize ubi 1 promoter in the binary vector pPZP201. A total of 320 plants showing GFP expression, derived from 45 calli, were selected and analyzed by Southern blot analysis. There was a 100% correlation between the GFP expression and the presence of the target gene, tlp, in these plants. Transgenic plants showing strong TLP expression were confirmed by Western blotting with antiserum specific for TLP. The transgene segregated in various ratios among progeny, which was confirmed by examining seedlings showing GFP fluorescence. The progeny also showed different copy numbers of transgenics. This report describes the successful use of GFP screening for efficient production of stably transformed sorghum plants without using antibiotics or herbicides as selection agents.Key words: Agrobacterium tumefaciens, green fluorescent protein (GFP), sorghum transformation.

Use of Green Fluorescent Protein To Tag and Investigate Gene Expression in Marine Bacteria

1998 ◽  
Vol 64 (7) ◽  
pp. 2554-2559 ◽  
Author(s):  
Serina Stretton ◽  
Somkiet Techkarnjanaruk ◽  
Alan M. McLennan ◽  
Amanda E. Goodman
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Marine Bacteria ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Bacterial Species ◽  
Epifluorescence Microscopy ◽  
Broad Host Range ◽  
Green Fluorescent

ABSTRACT Two broad-host-range vectors previously constructed for use in soil bacteria (A. G. Matthysse, S. Stretton, C. Dandie, N. C. McClure, and A. E. Goodman, FEMS Microbiol. Lett. 145:87–94, 1996) were assessed by epifluorescence microscopy for use in tagging three marine bacterial species. Expression of gfp could be visualized in Vibrio sp. strain S141 cells at uniform levels of intensity from either the lac or thenpt-2 promoter, whereas expression of gfp could be visualized in Psychrobacter sp. strain SW5H cells at various levels of intensity only from the npt-2 promoter. Green fluorescent protein (GFP) fluorescence was not detected in the third species, Pseudoalteromonas sp. strain S91, when thegfp gene was expressed from either promoter. A new mini-Tn10-kan-gfp transposon was constructed to investigate further the possibilities of fluorescence tagging of marine bacteria. Insertion of mini-Tn10-kan-gfp generated random stable mutants at high frequencies with all three marine species. With this transposon, strongly and weakly expressed S91 promoters were isolated. Visualization of GFP by epifluorescence microscopy was markedly reduced when S91 (mini-Tn10-kan-gfp) cells were grown in rich medium compared to that when cells were grown in minimal medium. Mini-Tn10-kan-gfp was used to create an S91 chitinase-negative, GFP-positive mutant. Expression of the chi-gfp fusion was induced in cells exposed toN′-acetylglucosamine or attached to chitin particles. By laser scanning confocal microscopy, biofilms consisting of microcolonies of chi-negative, GFP+ S91 cells were found to be localized several microns from a natural chitin substratum. Tagging bacterial strains with GFP enables visualization of, as well as monitoring of gene expression in, living single cells in situ and in real time.

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