Monitoring GFP‐operon fusion protein expression during high cell density cultivation of Escherichia coli using an on‐line optical sensor

DeLisa, Matthew P.; Li, Jincai; Rao, Govind; Weigand, William A.; Bentley, William E.

doi:10.1002/(sici)1097-0290(19991005)65:1<54::aid-bit7>3.3.co;2-i

Cited by 63 publications

(90 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…4b. Further support of this idea is seen in the responses to sodium acetate, serine hydroxamate, and hIL-2 overexpression, in that all three stressors have been observed to decrease the metabolic activity and/or capacity of E. coli (11,31,42). Therefore, it was not entirely surprising that the production of AI-2 was observed to decrease relative to the steady-state level following each of these perturbations.…”

Section: Discussionmentioning

confidence: 83%

“…Importantly, little is known about the attenuated induction of heterologous proteins commonly observed at high cell densities (11,19,50), where factors such as decreased growth rate, increased cell death, increased lysis, decreased metabolic activity, and segregation into viable but nonculturable cells can negatively impact reactor productivity (2,28,43). We have shown dramatically altered transcript levels of several stress-related genes at cell densities near 80 g (dry weight) liter Ϫ1 (20), and recent understanding of autoinduction suggests possible mechanisms that contribute to the altered metabolic and physiological state of cells grown to these cell densities.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Mapping Stress-Induced Changes in Autoinducer AI-2 Production in Chemostat-Cultivated Escherichia coli K-12

2001

Self Cite

View full text Add to dashboard Cite

Numerous gram-negative bacteria employ a cell-to-cell signaling mechanism, termed quorum sensing, for controlling gene expression in response to population density. Recently, this phenomenon has been discovered in Escherichia coli, and while pathogenic E. coli utilize quorum sensing to regulate pathogenesis (i.e., expression of virulence genes), the role of quorum sensing in nonpathogenic E. coli is less clear, and in particular, there is no information regarding the role of quorum sensing during the overexpression of recombinant proteins. The production of autoinducer AI-2, a signaling molecule employed by E. coli for intercellular communication, was studied in E. coli W3110 chemostat cultures using a Vibrio harveyi AI-2 reporter assay (M. G. Surrette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046-7050, 1998). Chemostat cultures enabled a study of AI-2 regulation through steady-state and transient responses to a variety of environmental stimuli. Results demonstrated that AI-2 levels increased with the steady-state culture growth rate. In addition, AI-2 increased following pulsed addition of glucose, Fe(III), NaCl, and dithiothreitol and decreased following aerobiosis, amino acid starvation, and isopropyl-␤-D-thiogalactopyranoside-induced expression of human interleukin-2 (hIL-2). In general, the AI-2 responses to several perturbations were indicative of a shift in metabolic activity or state of the cells induced by the individual stress. Because of our interest in the expression of heterologous proteins in E. coli, the transcription of four quorum-regulated genes and 20 stress genes was mapped during the transient response to induced expression of hIL-2. Significant regulatory overlap was revealed among several stress and starvation genes and known quorum-sensing genes.Synthesis and perception of a self-produced, freely diffusible signal molecule, termed autoinducer AI-2, by the gram-negative bacterium Escherichia coli is thought to regulate the expression of a variety of genes in response to population density. This process, termed autoinduction or quorum sensing, was first described in Vibrio fischeri (39), and similar autoregulatory mechanisms have since been reported in a wide range of bacteria, including Pseudomonas aeruginosa (34), Erwinia caratovora (5), and Agrobacterium tumefaciens (40), as well as E. coli (18,44,57). Although the existence of such a mechanism in E. coli has been uncovered, the genetic, physiological, and environmental factors that contribute to and regulate the quorum circuitry remain poorly defined.Evidence of intercellular communication in E. coli came with the discovery of a quorum-regulated transcriptional transactivator (SdiA) of the cell division genes in the ftsQAZ locus homologous to LuxR of V. fischeri (18, 44, 57) and a synthase protein (LuxS E.c. ) responsible for AI-2 signal molecule production (49). Further elucidation of native E. coli quorum circuit architecture resulted from the Vibrio harveyi cross-species activity assay of Surette and Bassler (47), which has g...

show abstract

Section: Discussionmentioning

confidence: 83%

mentioning

confidence: 99%

Mapping Stress-Induced Changes in Autoinducer AI-2 Production in Chemostat-Cultivated Escherichia coli K-12

2001

Self Cite

View full text Add to dashboard Cite

show abstract

“…Promoter activities were examined by measuring green fluorescent protein (GFP) production as an indirect, quantitative measurement of the transcriptional properties of cloned gfp (7,34). The culture-averaged fluorescence was measured by using a procedure described previously (18) and expressed as relative fluorescence units (RFU).…”

Section: Methodsmentioning

confidence: 99%

Directed Evolution of AraC for Improved Compatibility of Arabinose- and Lactose-Inducible Promoters

Lee¹,

Chou

Pfleger³

et al. 2007

Appl Environ Microbiol

View full text Add to dashboard Cite

Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (P BAD ) system that is more compatible with IPTG (isopropyl-␤-D-1-thiogalactopyranoside) induction of a lactose-inducible (P lac ) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.Recent advances in metabolic engineering and synthetic biology have increased the use of multiple genes (17) for various applications, such as the production of medicines (22,24) and the construction of complex genetic circuits (21, 30). Many genetic circuits, such as inverters (32), logic gates (8), pulse generators (4), band-pass filters, and oscillators (12), have been used to develop strains of bacteria that can communicate to form two-dimensional patterns, control their population density, and attack tumor cells in response to environmental cues (1,3,33). It is often necessary to introduce multiple genes and control the expression levels of those genes independently in order to accomplish these tasks. To date, a number of inducible promoters have been developed for Escherichia coli and other bacteria (2, 11, 16) which, in theory, should make it possible to independently control the expression of more than one gene. However, some pairs of promoters suffer from cross talk (an inducer of one promoter affects the expression from another promoter), making it difficult to simultaneously and independently control the expression levels of multiple genes. In contrast to electrical systems in which system interactions are well characterized and can be isolated from one another, it is often difficult or impossible to isolate biological components from activation or inhibition by other components in the cell.One example of cross talk involves a system containing two of the most useful promoters for gene expression in bacteria: the IPTG (isopropyl-␤-D-1-thiogalactopyranoside)-inducible lac promoter (P lac ) (15) and the arabinose-inducible araBAD promoter (P BAD ) (13, 29). The araBAD promoter system is regulated by the transcriptional regulator AraC. In the absence of arabinose, an AraC dimer creates a 210-base-pair DNA loop by contacting two widely separated half-sites (I 1 and O 2 ) on the DNA and represses transcription from P BAD (19,20,28). The binding of arabinose to AraC changes the position of the AraC dimer, causing the protein to preferentially bind different DNA-binding half-sites (I 1 and I 2 ). The change in configuration causes the dimer to release the DNA loop and allow transcription from P BAD (14,19,28).AraC is a 292-residue protein consisting of an N-terminal domain (residues 1 to 170) joined to a C-terminal DNA-b...

show abstract

“…The promoter-reporter plasmids pBAD24-gfp, pPro24-gfp, and pTrc99A-gfp were constructed by subcloning the PCR-amplified gfpuv gene encoding the UV-excitable green fluorescent protein (GFP) from p70GL into the multiple cloning site (MCS) of pBAD24, pPro24, and pTrc99A, respectively. GFP was used to provide an indirect, quantitative measurement of the transcriptional properties of the cloned gene (10,40).…”

Section: Methodsmentioning

confidence: 99%

A Propionate-Inducible Expression System for Enteric Bacteria

Lee

Keasling

2005

Appl Environ Microbiol

View full text Add to dashboard Cite

A series of new expression vectors (pPro) have been constructed for the regulated expression of genes in Escherichia coli. The pPro vectors contain the prpBCDE promoter (P prpB ) responsible for expression of the propionate catabolic genes (prpBCDE) and prpR encoding the positive regulator of this promoter. The efficiency and regulatory properties of the prpR-P prpB system were measured by placing the gene encoding the green fluorescent protein (gfp) under the control of the inducible P prpB of E. coli. This system provides homogenous expression in individual cells, highly regulatable expression over a wide range of propionate concentrations, and strong expression (maximal 1,500-fold induction) at high propionate concentrations. Since the prpBCDE promoter has CAP-dependent activation, the prpR-P prpB system exhibited negligible basal expression by addition of glucose to the medium.Inducible expression systems are essential molecular tools for production of recombinant proteins in cells, for synthesis and degradation of small molecules catalyzed by the enzymes expressed from the expression system, and for testing the function of unknown genes or proteins in cells. Although there are very good expression systems for high-level production of recombinant proteins, the more subtle approach of metabolic optimization has been hampered by the lack of appropriate systems for fine-tuning gene expression, and in particular expression systems that have homogeneous control in all cells of the culture.It has been reported that the all-or-none phenomenon occurs because the expression of the gene encoding the transporter for the inducer is controlled by the inducer itself (8,28,35). To alleviate the all-or-none phenomenon, control of expression of the gene encoding the transporter for the inducer itself was decoupled by placing the transporter gene under control of an inducer-independent promoter or using a synthetic inducer analogue that does not require active transport across the cell membrane (21,22). Since arabinose does not have a synthetic analogue that can freely diffuse across the cell membrane, the arabinose transporter gene was placed under control of a promoter that was not regulated by arabinose and homogenous expression from P BAD promoter was achieved (22). However, the araC-P BAD system requires genetically modified Escherichia coli strain as a host and is somewhat weaker than the P tac promoter (1, 2). On the other hand, the P lac promoter system has an effective gratuitous inducer (IPTG [isopropyl-␤-D-thiogalactopyranoside]) that can diffuse across the cell membrane without the aid of the lactose transporter (4); it has been shown that use of the gratuitous inducer IPTG eliminates the all-or-none response for the P tac and P trc promoters (20). However, IPTG is expensive (38), not digestible, and toxic (11). As such, IPTG contamination of recombinant protein products is undesirable (11).Other expression systems available include the tetracyclineinducible promoter P tet , the alkane-inducible promoter P alkB , ...

show abstract

Monitoring GFP‐operon fusion protein expression during high cell density cultivation of Escherichia coli using an on‐line optical sensor

Cited by 63 publications

References 0 publications

Mapping Stress-Induced Changes in Autoinducer AI-2 Production in Chemostat-Cultivated Escherichia coli K-12

Mapping Stress-Induced Changes in Autoinducer AI-2 Production in Chemostat-Cultivated Escherichia coli K-12

Directed Evolution of AraC for Improved Compatibility of Arabinose- and Lactose-Inducible Promoters

A Propionate-Inducible Expression System for Enteric Bacteria

Contact Info

Product

Resources

About