Coli Lac Operon Research Articles

The Inducible lac Operator-Repressor System Is Functional in Zebrafish Cells.

BackgroundZebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently available for editing genes and modifying gene expression in zebrafish, including RNAi, Cre/lox, and CRISPR-Cas9. However, the lac operator-repressor system, an E. coli lac operon component which has been adapted for use in many other species and is a valuable, flexible tool for inducible modulation of gene expression studies, has not been previously tested in zebrafish.ResultsHere we demonstrate that the lac operator-repressor system robustly decreases expression of firefly luciferase in cultured zebrafish fibroblast cells. Our work establishes the lac operator-repressor system as a promising tool for the manipulation of gene expression in whole zebrafish.ConclusionOur results lay the groundwork for the development of lac-based reporter assays in zebrafish, and adds to the tools available for investigating dynamic gene expression in embryogenesis. We believe this work will catalyze the development of new reporter assay systems to investigate uncharacterized regulatory elements and their cell-type specific activities.

The establishment process of lac operon model and the analysis of several teaching problems

Gene structure and expression regulation mechanism are the research hotspots and focus of modern life sciences. The lac operon is a cluster of genes through which Escherichia coli catabolizes lactose. It was first proposed by F. Jacob and J. Monod, who were also awarded the Nobel Prize in Physiology or Medicine in 1965 for their contributions. Thereafter, the lac operon has become the classic teaching case of the gene regulation mechanism in microbiology, genetics and molecular biology, and been highly valued by teachers and students alike. Although the conclusion is easy to follow and memorize, its rich connotation and esoteric reasoning has rendered it difficult to understand, neither is it easy for teachers to fully exploit the advantages of this teaching case. Therefore it is necessary to have an in-depth understanding of the genetic structure and working principle of the lac operon, especially the scientific background and thinking process through which scientists revealed these mysteries. In this paper, the historical discovery and analysis process of the E. coli lac operon was reviewed by following their footprints, listening to their analysis of experimental results. Based on the DNA sequences, the reasons for several unusual phenomena of lac operon expression were also discussed to exemplify the teaching value of the classic cases in genetics and molecular biology.

A Bacterial Adenylate Cyclase-Based Two-Hybrid System Compatible with Gateway® Cloning.

The bacterial adenylate cyclase two-hybrid system (BACTH) is a genetic approach used to test protein interactions in vivo in E. coli. This system takes advantage of the two catalytic domains of Bordetella pertussis adenylate cyclase (CyaA) toxin, which can be fused separately to proteins of interest. If the proteins of interest interact, then the adenylate cyclase domains will be brought in close proximity to each other, reconstituting cyclic AMP (cAMP) production. Interacting proteins can be both qualitatively and quantitatively assessed by the expression of chromosomal genes of the E. coli lac or mal operon, which are positively regulated by cAMP production. Because cAMP is diffusible, the proteins of interest do not need to interact near the transcriptional machinery. Consequently, both cytosolic and membrane protein-protein interactions can be tested. The BACTH system has recently been modified to be compatible with Gateway® recombinational cloning, BACTHGW. This chapter explains the principle of the BACTH, its Gateway® modified system, and details of the general procedure.

Construction of New Genetic Tools as Alternatives for Protein Overexpression in Escherichia coli and Pseudomonas aeruginosa.

Background:Pseudomonas protein expression in E. coli is known to be a setback due to significant genetic variation and absence of several genetic elements in E. coli for regulation and activation of Pseudomonas proteins. Modifications in promoter/repressor system and shuttle plasmid maintenance have made the expression of stable and active Pseudomonas protein possible in both Pseudomonas sp. and E. coli. Objectives: Construction of shuttle expression vectors for regulation and overexpression of Pseudomonas proteins in Pseudomonas sp. and E. coli. Materials and Methods:Pseudomonas-Escherichia shuttle expression vectors, pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo as well as E. coli expression vectors of pCon4 and pCon5 were constructed from pUCP19-, pSS213-, pSTBlue-1- and pPICZαA-based vectors. Protein overexpression was measured using elastase strain K as passenger enzyme in elastinolytic activity assay. Results: The integration of two series of IPTG inducible expression cassettes in pCon2(3), pCon2(3)-Kan and pCon2(3)-Zeo, each carrying an E. coli lac-operon based promoter, Plac, and a tightly regulated T7(A1/O4/O3) promoter/repressor system was performed to facilitate overexpression study of the organic solvent-tolerant elastase strain K. These constructs have demonstrated an elastinolytic fold of as high as 1464.4 % in comparison to other published constructs. pCon4 and pCon5, on the other hand, are series of pCon2(3)-derived vectors harboring expression cassettes controlled by PT7(A1/O4/O3) promoter, which conferred tight regulation and repression of basal expression due to existence of respective double operator sites, O3 and O4, and lacIq. Conclusions: The constructs offered remarkable assistance for overexpression of heterogeneous genes in Pseudomonas sp. and E. coli for downstream applications such as in industries and structural biology study.

Occlusion of the Ribosome Binding Site Connects the Translational Initiation Frequency, mRNA Stability and Premature Transcription Termination

Protein production is controlled by ribosome binding to the messenger RNA (mRNA), quantified in part by the binding affinity between the ribosome and the ribosome binding sequence on the mRNA. Using the E. coli lac operon as model, Ringquist et al. (1992) found a more than 1,000-fold difference in protein yield when varying the Shine-Dalgarno sequence and its distance to the translation start site. Their proposed model accounted for this large variation by only a variation in the binding affinity and the subsequent initiation rate. Here we demonstrate that the decrease in protein yield with weaker ribosome binding sites in addition is caused by a decreased mRNA stability, and by an increased rate of premature transcription termination. Using different ribosome binding site sequences of the E. coli lacZ gene, we found that an approximately 100-fold span in protein expression could be subdivided into three mechanisms that each affected expression 3- to 6-fold. Our experiments is consistent with a two-step ribosome initiation model, in which occlusion of the initial part of the mRNA by a ribosome simultaneously protects the mRNA from both premature transcription termination and degradation: The premature termination we suggest is coupled to the absence of occlusion that allows binding of transcription termination factor, possibly Rho. The mRNA stability is explained by occlusion that prevents binding of the degrading enzymes. In our proposed scenario, a mRNA with lower translation initiation rate would at the same time be “hit” by an increased premature termination and a shorter life-time. Our model further suggests that the transcription from most if not all natural promoters is substantially influenced by premature termination.

In Pursuit of Genes of Glucose Metabolism

If you don't know where you're going, you might wind up someplace else—Yogi Berra My professional journey, once I accepted the fact that I would not play first base for the Chicago Cubs, was not accomplished in a straight line. I entered medical school intending to become a family physician but soon became interested in research. I took an internal medicine residency at the University of California, San Francisco (UCSF), then a long research fellowship in the nascent field of molecular biology at the National Institutes of Health (NIH), and followed that with training in clinical endocrinology at the University of Wisconsin. I spent the next fifteen years dividing my time between the clinic and laboratory at the University of Iowa. Finally, I decided to concentrate on research and spent the next twenty-three years at Vanderbilt University. For the past eleven years, I have been at the University of Iowa, where I helped establish a new diabetes research center. My research journey also had several different directions. Upon reflection, I guess I ended up “someplace else” by choice, and it certainly has been an interesting trip. It is one I unreservedly recommend.

Inducible genetic system for the axolotl

Transgenesis promises a powerful means for assessing gene function during amphibian limb regeneration. This approach is complicated, however, by the need for embryonic appendage development to proceed unimpeded despite the genetic alterations one wishes to test later in the context of regeneration. Achieving conditional gene regulation in this amphibian has not proved to be as straightforward as in many other systems. In this report we describe a unique method for obtaining temporal control over exogenous gene expression in the axolotl. Based on technology derived from the Escherichia coli Lac operon, uninduced transgenes are kept in a repressed state by the binding of constitutively expressed Lac repressor protein (LacI) to operator sequences within the expression construct. Addition of a lactose analog, IPTG, to the swimming water of the axolotl is sufficient for the sugar to be taken up by cells, where it binds the LacI protein, thereby inducing expression of the repressed gene. We use this system to demonstrate an in vivo role for thrombospondin-4 in limb regeneration. This inducible system will allow for systematic analysis of phenotypes at defined developmental or regenerative time points. The tight regulation and robustness of gene induction combined with the simplicity of this strategy will prove invaluable for studying many aspects of axolotl biology.

Direct measurements of IPTG enable analysis of the induction behavior of E. coli in high cell density cultures

BackgroundThe E. coli lac operon and its components have been studied for decades, and lac-derived systems are widely used for recombinant protein production. However, lac operon dynamics and induction behavior remain the paradigm of gene regulation. Recently, an HPLC-MS-based method to quantify IPTG in the medium and inside the biomass has been established, and this tool may be useful to uncover the lack of knowledge and allow optimization of biotechnological processes.ResultsThe results obtained from the study of IPTG distribution profiles in fed-batch, high cell density cultures allowed discrimination between two different depletion patterns of an inducer from the medium to the biomass in E. coli-expressing rhamnulose-1-phosphate aldolase (RhuA). Moreover, we could demonstrate that active transport mediates the uptake of this gratuitous inducer. Additionally, we could study the induction behaviors of this expression system by taking into account the biomass concentration at the induction time.ConclusionsIn the bistable range, partial induction occurred, which led to intermediate levels of RhuA activity. There was a direct relationship between the initial inducer concentrations and the initial inducer transport rate together with the specific activity. A majority of the inducer remains in the medium to reach equilibrium with the intracellular level. The intracellular inducer accumulation was a further evidence of bistability of the lac operon.

Genetically manipulated mice: a powerful tool with unsuspected caveats

Although genetic manipulations in mice have provided a powerful tool for investigating gene function in vivo, recent studies have uncovered a number of developmental phenomena that complicate the attribution of phenotype to the specific genetic change. A more realistic approach has been to modulate gene expression and function in a temporal and tissue-specific manner. The most common of these methods, the CreLoxP and tetracycline response systems, are surveyed here and their recently identified shortcomings discussed, along with a less well known system based on the E. coli lac operon and modified for use in mammals. The potential for further complications in interpretation due to hitherto unexpected epigenetic effects involving transfer of RNA or protein in oocytes or sperm is also explored. Given these problems we reiterate the necessity for the use of completely reversible methods that will allow each experimental group of animals to act as their own control. Using these methods with a number of specific modifications to eliminate non-specific effects from random insertion sites and inducer molecules, the full potential of genetic manipulation studies should be realized.

Modeling the Lac repressor-operator assembly: The influence of DNA looping on Lac repressor conformation

Repression of transcription of the Escherichia coli Lac operon by the Lac repressor (LacR) is accompanied by the simultaneous binding of LacR to two operators and the formation of a DNA loop. A recently developed theory of sequence-dependent DNA elasticity enables one to relate the fine structure of the LacR-DNA complex to a wide range of heretofore-unconnected experimental observations. Here, that theory is used to calculate the configuration and free energy of the DNA loop as a function of its length and base-pair sequence, its linking number, and the end conditions imposed by the LacR tetramer. The tetramer can assume two types of conformations. Whereas a rigid V-shaped structure is observed in the crystal, EM images show extended forms in which two dimer subunits are flexibly joined. Upon comparing our computed loop configurations with published experimental observations of permanganate sensitivities, DNase I cutting patterns, and loop stabilities, we conclude that linear DNA segments of short-to-medium chain length (50-180 bp) give rise to loops with the extended form of LacR and that loops formed within negatively supercoiled plasmids induce the V-shaped structure.

MODELLING FOR CONTROL: UNDERSTANDING ROLE AND FUNCTION OF REGULATORY NETWORKS IN MICROORGANISMS

Microorganisms maintain a constant monitoring of extra-cellular physicochemical conditions in order to respond and modify their gene expression patterns accordingly. Modelling plays an essential role in developing an understanding of the regulatory networks in microorganisms. Means-end analysis, also called functional modelling, is proposed as a methodology to model regulatory networks. A modelling example for the E. coli lac operon illustrates that the methodology allows breaking down complex regulatory networks into elementary building blocks. The proposed modelling formalism enables a straightforward communication between systems engineers and biologists on the mechanisms underlying complex regulatory functions.

Genome organization and reorganization in evolution: formatting for computation and function.

This volume deals with the role of epigenetics in life and evolution. The most dynamic forms of functional genome formatting involve DNA interacting with cellular complexes that do not alter sequence information. Such important epigenetic phenomena are the main subjects of other articles in this volume. This article focuses on the long-lived form of genome formatting that lies within the DNA sequence itself. I argue for a computational view of genome function as the long-term information storage organelle of each cell. Structural formatting consists of organizing various signals and coding sequences into computationally ready systems facilitating genome expression and genome transmission. The basic features of genome organization can be understood by examining the E. coli lac operon as a paradigmatic genomic system. Multiple systems are connected through distributed signals and repetitive DNA to form higher-order genome system architectures. Molecular discoveries about mechanisms of DNA restructuring show that cells possess the natural genetic engineering functions necessary for evolutionary change by rearranging genomic components and reorganizing system architectures. The concepts of cellular computation and decision-making, genome system architecture, and natural genetic engineering combine to provide a new way of framing evolutionary theories and understanding genome sequence information.

Integrative modeling of gene expression and metabolism with E-CELL System

A gene-expression model for an E-CELL simulation was constructed, and the simulation was carried out. The model was integrated with a metabolism model of the self-sustaining virtual cell model which was constructed based on aMycoplasma genitalium cell. Details of the model structure and the results of the simulation are shown. AnEscherichia coli lac-operon model was also constructed by integrating and abstracting the gene-expression model. Wherever possible, experimental parameters were used to construct the model

Intracellular cyclic AMP concentration is decreased in Salmonella typhimurium fur mutants.

It is known that the Fur protein negatively regulates iron-uptake systems in different bacterial species, including Salmonella typhimurium. In this study it has been shown that the intracellular concentration of cyclic AMP (cAMP) is lower in a knockout S. typhimurium fur mutant than in the wild-type strain. According to this, the expression of two cAMP-regulated genes, such as pepE (encoding an alpha-aspartyl dipeptidase) and the Escherichia coli lac operon, is decreased in S. typhimurium fur cells in comparison with wild-type cells. Introduction of an additional mutation in cpdA, encoding a cyclic 3',5'-cAMP phosphodiesterase, recovers wild-type intracellular cAMP concentration in the S. typhimurium fur mutant. Likewise, expression of pepE and the E. coli lac operon was the same in the S. typhimurium fur cpdA double mutant and the wild-type strain. Moreover, these results also demonstrate that the S. typhimurium Fur protein positively regulates the expression of the flhD master operon governing the flagellar regulon. This positive control must be mediated by binding of the S. typhimurium Fur protein to the flhD promoter as indicated by the fact that this promoter tests positive in a Fur titration assay.

Signal and noise in bridging PCR

BackgroundIn a variant of the standard PCR reaction termed bridging, or jumping, PCR the primer-bound sequences are originally on separate template molecules. Bridging can occur if, and only if, the templates contain a region of sequence similarity. A 3' end of synthesis in one round of synthesis that terminates in this region of similarity can prime on the other. In principle, Bridging PCR (BPCR) can detect a subpopulation of one template that terminates synthesis in the region of sequence shared by the other template. This study considers the sensitivity and noise of BPCR as a quantitative assay for backbone interruptions. Bridging synthesis is also important to some methods for computing with DNA.ResultsIn this study, BPCR was tested over a 328 base pair segment of the E. coli lac operon and a signal to noise ratio (S/N) of approximately 10 was obtained under normal PCR conditions with Taq polymerase. With special precautions in the case of Taq or by using the Stoffel fragment the S/N was improved to 100, i.e. 1 part of cut input DNA yielded the same output as 100 parts of intact input DNA.ConclusionsIn the E. coli lac operator region studied here, depending on details of protocol, between 3 and 30% per kilobase of final PCR product resulted from bridging. Other systems are expected to differ in the proportion of product that is bridged consequent to PCR protocol and the sequence analyzed. In many cases physical bridging during PCR will have no informational consequence because the bridged templates are of identical sequence, but in a number of special cases bridging creates, or, destroys, information.

An experimental and theoretical study of the inhibition of Escherichia coli lac operon gene expression by antigene oligonucleotides.

Previously, we have developed a genetically structured mathematical model to describe the inhibition of Escherichia coli lac operon gene expression by antigene oligos. Our model predicted that antigene oligos targeted to the operator region of the lac operon would have a significant inhibitory effect on beta-galactosidase production. In this investigation, the E. coli lac operon gene expression in the presence of antigene oligos was studied experimentally. A 21-mer oligo, which was designed to form a triplex with the operator, was found to be able to specifically inhibit beta-galactosidase production in a dose-dependent manner. In contrast to the 21-mer triplex-forming oligonucleotide (TFO), several control oligos showed no inhibitory effect. The ineffectiveness of the various control oligos, along with the fact that the 21-mer oligo has no homology sequence with lacZYA, and no mRNA is transcribed from the operator, suggests that the 21-mer oligo inhibits target gene expression by an antigene mechanism. To simulate the kinetics of lac operon gene expression in the presence of antigene oligos, a genetically structured kinetic model, which includes transport of oligo into the cell, growth of bacteria cells, and lac operon gene expression, was developed. Predictions of the kinetic model fit the experimental data quite well after adjustment of the value of the oligonucleotide transport rate constant (9.0 x 10(-)(3) min(-)(1)) and oligo binding affinity constant (1.05 x 10(6) M(-)(1)). Our values for these two adjusted parameters are in the range of reported literature values.

SOS Mutator DNA Polymerase IV Functions in Adaptive Mutation and Not Adaptive Amplification

Adaptive point mutation and amplification are induced responses to environmental stress, promoting genetic changes that can enhance survival. A specialized adaptive mutation mechanism has been documented in one Escherichia coli assay, but its enzymatic basis remained unclear. We report that the SOS-inducible, error-prone DNA polymerase (pol) IV, encoded by dinB, is required for adaptive point mutation in the E. coli lac operon. A nonpolar dinB mutation reduces adaptive mutation frequencies by 85% but does not affect adaptive amplification, growth-dependent mutation, or survival after oxidative or UV damage. We show that pol IV, together with the major replicase, pol III, can account for all adaptive point mutations at lac. The results identify a role for pol IV in inducible genetic change.

The inhibition of Escherichia coli lac operon gene expression by antigene oligonucleotides-mathematical modeling.

Gene transcription is regulated by transcription factors that can bind to specific regions on DNA. Antigene oligonucleotides (oligos) can bind to specific regions on DNA and form a triplex with the double-stranded DNA. The triplex can competitively inhibit the binding of transcription factors and, as a result, transcription can be inhibited. A genetically structured model has been developed to quantitatively describe the inhibition of the Escherichia coli lac operon gene expression by triplex-forming oligos. The model predicts that the effect of triplex-forming oligos on the lac operon gene expression depends on their target sites. Oligonucleotides targeted to the operator are much more effective than those targeted to other regulatory sites on the lac operon. In some cases, the effect of oligo binding is similar to that of a mutation in the lac operon. The model provides insight as to the specific binding site to be targeted to achieve the most effective inhibition of gene expression. The model is also capable of predicting the oligo concentration needed to inhibit gene expression, which is in general agreement with results reported by other investigators.

Stringent regulation of human growth hormone expression in cultured murine C2C12 myoblasts by theE. coli lac repressor

Gene transfer techniques can be used to encode the production of a polypeptide product, such as human growth hormone (hGH), that is missing in an acquired or inherited disease state such as growth hormone deficiency. In one model system, engineered C2C12 myoblasts are injected intramuscularly into a mouse and subsequently secrete hGH into the circulation. In this regard, a gene-expression regulatory system that functions in myoblasts would be of interest. We demonstrate that the Escherichia coli ldc operon system can be used to stringently regulate the expression of hGH in engineered C2C12 myoblasts in tissue culture. A DNA segment encoding hGH was linked to a DNA segment containing an SV40 enhancer and promoter. The latter components were positioned between two synthetic lac operators. Lac repressor expression was driven by a simian cytomegalovirus promoter. In transient co-transfection assays, hGH expression from cultured C2C12 myoblasts could be modulated up to 60-fold (P = 0.002) with the inducing agent, isopropyl-beta-D-thiogalactoside (IPTG). In the absence of IPTG, hGH expression was almost fully repressed. These results show that the components of the E. coli lac operon provide a stringent regulatory system for use in myoblasts. The system might prove to be useful for the regulation of transferred genes in animals.

Occurrence of oligopurine.oligopyrimidine tracts in eukaryotic and prokaryotic genes

A program to analyse the length and frequency distribution of specific base tracts in genomic sequences is described. The frequency of oligopurine.oligopyrimidine tracts (R.Y. tracts) in a data base of 163 transcribed genes is analysed and compared. The complete genomes of SV40 virus, N. tobacum chloroplast, yeast 2 micron plasmid, bacteriophage lambda, plasmid pBR322 and the E. coli lac operon are also analyzed. A highly significant overrepresentation of oligopurine and oligopyrimidine tracts is observed in all eukaryotic genes examined, as well as in the chloroplast genome. The overrepresentation is evident in all gene subregions of the chloroplast, in the following order: intergenic regions, 3' downstream and 5' upstream (promoter), 5' and 3' untranslated, introns and coding regions. In genes coding for basic proteins, oligopurine rather than oligopyrimidine tracts are found on the coding stand. In prokaryotic genes only the longest R.Y. tracts (greater than or equal to 12) are found in excess, and are concentrated near regulatory regions. While a structural role for R.Y. tracts is most likely in intergenic regions, a functional role, as initiation sites for strand separation, is proposed for regulatory gene regions.