GFPuv Expression Research Articles

\(\textit{n}\)-alkanol stress-induced cell envelope injury of \(σ^{E}\) promoter in \(\textit{Escherichia coli}\)

To characterize the cellular stress by n-alkanols with different alkyl chain lengths in Escherichia coli, we investigated how n-alkanols damage cell envelope permeability and whether they enhance the promoter activity of the envelope stress response regulator, σE, by using variants of green fluorescent protein (GFP). By using E. coli cells having GFPuv expressing and localizing in the cytoplasm, the inner membrane, and the periplasm, after exposure to n-alkanols, the fluorescent intensity of GFPuv released from cells was examined. Our data showed that at the similar levels of cell death of about 90–97%, ethanol, a short-chain alkanol, at a concentration of 20% damaged the outer membrane more greatly than the inner membrane, whereas a longer-chain alkanol of pentanol at a concentration of 1.125% damaged both of the outer and inner membranes. Then we investigated the envelope stress response to n-alkanols by σE factor by ratiometric analysis of rpoE promoter activity for the downstream GFPuv expression referenced to that of housekeeping sigma 70 (σ70 ) recognizing lacUV5 promoter for red fluorescent protein (RFP) expression. The results indicated that the relative activity of rpoE promoter by pentanol was much greater than that of ethanol. The degree of its sensitization by rpoE deficiency was much more remarkable for cells treated with pentanol than for those with ethanol. The results suggest that the response of the σE plays a significant role in the membrane integrity and survival of E. coli cells treated with n-alkanols depending on the alkyl chain length of the molecule.

Boosting Auto-Induction of Recombinant Proteins in Escherichia coli with Glucose and Lactose Additives.

BackgroundAuto-induction is a convenient way to produce recombinant proteins without inducer addition using lac operon-controlled Escherichia coli expression systems. Auto-induction can occur unintentionally using a complex culture medium prepared by mixing culture substrates. The differences in culture substrates sometimes lead to variations in the induction level.ObjectivesIn this study, we investigated the feasibility of using glucose and lactose as boosters of auto-induction with a complex culture medium.MethodsFirst, auto-induction levels were assessed by quantifying recombinant GFPuv expression under the control of the T7 lac promoter. Effectiveness of the additive-containing medium was examined using ovine angiotensinogen (tac promoter-based expression) and Thermus thermophilus manganese-catalase (T7 lac promoter-based expression).ResultsAuto-induced GFPuv expression was observed with the enzymatic protein digest Polypepton, but not with another digest tryptone. Regardless of the type of protein digest, supplementing Terrific Broth medium with glucose (at a final concentration of 2.9 g/L) and lactose (at a final concentration of 7.6 g/L) was successful in obtaining an induction level similar to that achieved with a commercially available auto-induction medium. The two recombinant proteins were produced in milligram quantity of purified protein per liter of culture.ConclusionThe medium composition shown in this study would be practically useful for attaining reliable auto-induction for E. coli-based recombinant protein production.

Microbioreactor with Photodiode Detection for Monitoring Intracellular GFPUV Expression in E. coli

Microbioreactor with Photodiode Detection for Monitoring Intracellular GFPUV Expression in E. coli

The development and characterization of an exogenous green-light-regulated gene expression system in marine cyanobacteria.

A green-light-regulated gene expression system derived from Synechocystis sp. PCC 6803 was constructed and introduced into the marine cyanobacterial strain Synechococcus sp. NKBG 15041c. The regulation system was evaluated using gfp uv as a reporter gene under red-light illumination and under simultaneous red- and green-light illumination. Expression of the reporter gene was effectively repressed under red-light illumination and increased over 10-fold by illuminating with green light. Control vectors missing either the ccaS sensor histidine kinase gene or the ccaR response regulator gene showed no detectable induction of GFPuv expression. Green-light induction of gfp uv expression was further confirmed by quantitative reverse transcription PCR. The constructed system was effective at regulating the recombinant expression of a target gene using green light in a marine cyanobacterial strain that does not naturally possess such a green-light regulation system. Thus, constructed green-light-regulated gene expression system may be used as a core platform technology for the development of marine cyanobacterial strains in which bioprocesses will be regulated by light.

Microbioreactor with Photodiode Detection for Monitoring Intracellular GFPUV Expression in E. coli

Microbioreactor with Photodiode Detection for Monitoring Intracellular GFPUV Expression in E. coli

Construction of a reporter system for Lactobacillus sp. using the gfpuv gene

Lactobacilli are bacteria commonly associated with the gastrointestinal tract of animals and humans. They are able to produce antimicrobial substances such as bacteriocins, lactic acid and hydrogen peroxide, the factors which have been shown to be beneficial for controlling overgrowth of potentially pathogenic bacteria. For this reason lactobacilli are often applied in probiotics. The aim of present study was to construct a reporter strain based on the gfpuv expression system which can bee used as a strain with the ability to colonize the intestinal tract in future experiments.

Microbial nar-GFP cell sensors reveal oxygen limitations in highly agitated and aerated laboratory-scale fermentors

BackgroundSmall-scale microbial fermentations are often assumed to be homogeneous, and oxygen limitation due to inadequate micromixing is often overlooked as a potential problem. To assess the relative degree of micromixing, and hence propensity for oxygen limitation, a new cellular oxygen sensor has been developed. The oxygen responsive E. coli nitrate reductase (nar) promoter was used to construct an oxygen reporter plasmid (pNar-GFPuv) which allows cell-based reporting of oxygen limitation. Because there are greater than 109 cells in a fermentor, one can outfit a vessel with more than 109 sensors. Our concept was tested in high density, lab-scale (5 L), fed-batch, E. coli fermentations operated with varied mixing efficiency – one verses four impellers.ResultsIn both cases, bioreactors were maintained identically at greater than 80% dissolved oxygen (DO) during batch phase and at approximately 20% DO during fed-batch phase. Trends for glucose consumption, biomass and DO showed nearly identical behavior. However, fermentations with only one impeller showed significantly higher GFPuv expression than those with four, indicating a higher degree of fluid segregation sufficient for cellular oxygen deprivation. As the characteristic time for GFPuv expression (approx 90 min.) is much larger than that for mixing (approx 10 s), increased specific fluorescence represents an averaged effect of oxygen limitation over time and by natural extension, over space.ConclusionThus, the pNar-GFPuv plasmid enabled bioreactor-wide oxygen sensing in that bacterial cells served as individual recirculating sensors integrating their responses over space and time. We envision cell-based oxygen sensors may find utility in a wide variety of bioprocessing applications.

Determination of Geno- and Cytotoxicity of Groundwater and Sediments Using the Recombinant SWITCH Test*

The recombinant Salmonella typhimurium TA1535 strain carrying the SWITCH plasmid (combined construct of the SOS-Lux plasmid pPLS-1 and the LAC-Fluoro plasmid pGFPuv: pSWITCH) was treated with control substances for genotoxic (2-aminoanthracene) and cytotoxic (aureomycin) potency as well as with 18 environmental samples (groundwater, river water, sediments) provided at the SENSPOL Technical Meeting on Problems Related to Diffuse Pollution Sources (Characterization of Sediment, Dredged Material, and Groundwater) organized by the Federal Institute of Hydrology in Koblenz, Germany, during late October 2003. For metabolic activation the samples were treated with S9 mix (5% S9 fraction in cofactor mix, Aroclor 1254-induced rat liver microsomes, Moltox Inc., Mol, Belgium). Simultaneously determined cytotoxicity and genotoxicity data were derived through the Multilabel Counter 1420 Victor2 (PerkinElmer, Boston, MA, USA), by sequential measurement of luminescence, absorbance, and fluorescence. The newly designed SWITCH test, as it was applied at the Koblenz meeting, displays a comparable sensitivity for test samples with known cyto- and genotoxic potential. Groundwater samples from the wells at the former gas plant site Kiel Canal and the agricultural area at Niederwerth expressed neither genotoxic nor cytotoxic responses of the bacteria for both metabolic conditions (+/−S9). Spiked groundwater samples from the Niederwerth well BW1 and the Urmitz well U12 located on the river Rhine were identified to be positive in terms of genotoxicity for the direct and the metabolic approach. Samples from the lake Tiefer See in the city of Potsdam showed a reduction of GFPuv expression as an indication for cytotoxicity, while luminescence output of incubated bacteria remained unaffected. This reflects the well-known presence of contaminations (especially cyanides) in the lake sediment as well as in the acetonic extract. The results obtained at the SENSPOL Technical Meeting show the SWITCH test to be of major relevance not only for the analysis of chemicals under laboratory conditions but also for environmental samples polluted by diffuse industrial sources.

Demonstration of microbial transport into the capillary fringe via advection from below the water table

A laboratory experiment is used to provide preliminary evidence that microbes can be advected into the capillary fringe from the region below the water table under steady flow conditions. A flow cell was packed so as to contain both a region for which pore-water pressure was greater than atmospheric pressure (termed ‘below the water table’) and a region, where the pore-water pressure was less than atmospheric with the pores essentially saturated with water (termed the ‘capillary fringe’). Steady flow was then established through maintaining a hydraulic gradient across the medium. Green fluorescent protein (GFP) transformed bacteria ( E. coli JM109) were used to visualize migration of bacteria from below the water table into the capillary fringe. These transformed bacteria fluoresce brightly and readily when excited by standard UV light (395 nm) in 100 mL LB medium with 100 μg/mL ampicillin. The concentrations of bacterial inoculum and oxygen were adjusted to ensure GFPuv expression at a large scale. Results demonstrated that microbes can move into the capillary fringe from below the water table under horizontal hydraulic gradients. Motion from the capillary fringe into the region below the water table was also observed as was the absence of advection through regions of entrapped air below the water table.

English

The recombinant green fluorescent protein (GFPuv) was expressed by transformed cells of Escherichia coli DH5-a grown in LB/amp broth at 37oC, for 8 h and 24 h. To evaluate the effectiveness of different parameters to improve the expression of GFPuv by E. coli, four variable culturing conditions were set up for assays by a fractional factorial (24-1) design at two levels: (i) the effect of storing (24-48 h) the seeded broth at 4oC prior to incubation at 37oC; (ii) the effect of agitation speed (100-200 rpm); (iii) the final concentration (0.05-0.5 mM) of IPTG (isopropyl–b-D-thiogalactopyranoside); and (iv) the addition of IPTG at set cell densities (OD660 0.01-0.8).  GFPuv was extracted from cells by the three phase partitioning method (TPP) and further purified with a methyl HIC column. The cultures grown at 37oC/24 h provided the highest yields of GFPuv under the conditions: (i) pre-storage at 4oC/24 h; (ii) agitation speed at 100 rpm; (iii) 0.5 mM IPTG; (iv) IPTG addition at OD660=~0.01. On the other hand, at 37oC/ 8 h, GFPuv expression was dependent upon agitation of broth cultures at 200 rpm and the IPTG addition at the beginning of the growth exponential phase.    Key words: Green fluorescent protein (GFPuv), Escherichia coli DH5-a, growth kinetic parameters, expressed GFPuv kinetic parameters, three phase partitioning extraction (TPP).

Evaluation of recombinant green fluorescent protein, under various culture conditions and purification with HiTrap hydrophobic interaction chromatography resins.

To determine the influence of various culture conditions, transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were grown in nine cultures with four variable conditions (storage of inoculated broth at 4 degrees C prior to incubation, agitation speed, isopropyl-beta-D-thiogalactopyranoside [IPTG] concentration, and induction time). The pelleted cells were resuspended in extraction buffer and subjected to the three-phase partitioning (TPP) extraction method. To determine the most appropriate purification resin, protein extracts were eluted through one of four types of HiTrap hydrophobic interaction chromatography (HIC) columns prepacked with methyl, butyl, octyl, or phenyl resins and analyzed further on a 12% sodium dodecylsulfate polyacrylamide gel. With Coomassie staining, a single band between 27 (standard GFPuv) and 29 kDa (molecular weight standard) was visualized for every HIC column sample. TPP extraction with HIC elution provided about 90% of the GFPuv recovered and eight-fold GFPuv enrichment related to the specific mass. Rotary speed and IPTG concentration showed, respectively, greater negative and positive influences on GFPuv expression at the beginning of the logarithmic phase for the set culture conditions (37 degrees C, 24-h incubation).

The effect of cell cycle on GFPuv gene expression in the baculovirus expression system

The effects of cell cycle on recombinant protein production and infection yield in the baculovirus-insect cell expression system (BES) were investigated. When, at any cell cycle phase, the host cell was infected by baculovirus, the cell cycle was finally arrested at the S or G 2/M phase with 4n DNA. In the case of G 1 or S phase-infection, cell cycle of virus-infected cells began to be arrested at S phase from 8 h post-infection or at G 2/M phase from 4 h post-infection, respectively; while, in the case of M phase-infection, cell cycle was arrested at S phase after 12 h post-infection. When the host cell was infected at the G 1 phase, average intracellular GFPuv fluorescence intensity was 1.3-fold higher than that at G 2/M phase at 24 h post-infection. The GFPuv expression corresponded to the profile of the G 1 cell cycle in the BES. Infection yield was measured by detection of intracellular DNA binding protein using immunohistochemical method within 7 h post-infection. The infection yield at G 1 or S phase-infection was 1.5–1.8-fold higher than that at G 2/M phase-infection.