lacZ Research Articles

Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector

In this study, a cryptic plasmid pOfk55 from Legionella pneumophila was isolated and characterized. pOfk55 comprised 2584bp with a GC content of 37.3% and contained three putative open reading frames (ORFs). orf1 encoded a protein of 195 amino acids and the putative protein shared 39% sequence identity with a putative plasmid replication protein RepL. ORF1 was needed for replication in L. pneumophila but pOfk55 did not replicate in Escherichia coli. orf2 and orf3 encoded putative hypothetical proteins of 114 amino acids and 78 amino acids, respectively, but the functions of the putative proteins ORF2 and OFR3 are not clear. The transfer mechanism for pOfk55 was independent on the type IVB secretion system in the original host. A L. pneumophila–E. coli shuttle vector, pNT562 (5058bp, KmR), was constructed by In-Fusion Cloning of pOfk55 with a kanamycin-resistance gene from pUTmini-Tn5Km and the origin of replication from pBluescript SK(+) (pNT561). Multiple cloning sites from pBluescript SK(+) as well as the tac promoter region and lacI gene from pAM239-GFP were inserted into pNT561 to construct pNT562. The transformation efficiency of pNT562 in L. pneumophila strains ranged from 1.6×101 to 1.0×105CFU/ng. The relative number of pNT562 was estimated at 5.7±1.0 copies and 73.6% of cells maintained the plasmid after 1week in liquid culture without kanamycin. A green fluorescent protein (GFP) expression vector, pNT563, was constructed by ligating pNT562 with the gfpmut3 gene from pAM239-GFP. pNT563 was introduced into L. pneumophila Lp02 and E. coli DH5α, and both strains expressed GFP successfully. These results suggest that the shuttle vector is useful for genetic studies in L. pneumophila.

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313.

Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a β-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313 lacI genes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pH fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5- to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ΔglyR1 strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ΔglyR2 strain, and α-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ΔglyR3 strain, upregulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters.IMPORTANCE Understanding C. thermocellum gene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specific C. thermocellum LacI regulons. The identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum.

Engineering DNA Looping in E. Coli

DNA looping plays an important role in many cellular functions including replication, transcription, DNA packaging, and recombination. DNA repression loop formation in prokaryotes acts to regulate transcription in response to a changing environment. One example of gene control by DNA loop formation occurs in the well-studied lac operon. E. coli lac loop formation occurs when LacI, a tetrameric protein, simultaneously binds two DNA operators within close proximity, resulting in a DNA loop. This structure represses transcription of downstream operon genes. We are curious if repression loop formation can be targeted in vivo. Our goal is an engineered protein fusion with both sequence specific DNA binding function and chemically induced dimerization (CID) function. The chemically dimerized protein would have the ability to bind the DNA in a bidentate manner, analogous to LacI, resulting in DNA looping. Targeted DNA loop formation could act in gene repression and might be tunable by the concentration of CID agent. Using designed transcription activator-like effector (TALE) proteins allows creation of DNA binding proteins with arbitrary DNA sequence specificity. TALEs have been used for in vivo genome-engineering applications. FK506 binding protein (FKBP) provides a well-studied protein domain whose ability to undergo CID is exploited in our designs. We utilize our established lac promoter looping system to assess TALE-FKBP protein fusions targeted to lac operators.

Construction of a BioBrick™ compatible vector system for Rhodococcus

Throughout the past decade, the field of synthetic biology has grown rapidly. By using assembly platforms such as BioBricks™, scientists can quickly and easily build gene circuits or multi-step pathways. One limitation, however, is that most of these parts were designed and characterized with Escherichia coli as the target chassis. As a consequence, there exists a lack of standardized and well characterized or BioBrick™ compatible plasmid backbones that replicate in other potential non-model chassis organisms. The Gram-positive bacteria of the genus Rhodococcus represent an interesting chassis for biotechnological applications due to their tremendous metabolic capabilities. In this report we describe our progress toward developing a BioBrick™ compatible plasmid system for Rhodococcus. We demonstrate its utility for heterologous protein expression through flow cytometric analysis of the lac promoter in the oleaginous strain Rhodococcus opacus PD630.

Screening through the PLICable promoter toolbox enhances protein production in Escherichia coli.

Escherichia coli is a common host for recombinant protein production in which production titers are highly dependent on the employed expression system. Promoters are thereby a key element to control gene expression levels. In this study, a novel PLICable promoter toolbox was developed which enables in a single cloning step and after a screening experiment to identify out of ten IPTG-inducible promoters (T7, A3, lpp, tac, pac, Sp6, lac, npr, trc and syn) the most suitable one for high level protein production. The target gene is cloned under the control of different promoters in a single and efficient cloning step using the ligase-free cloning method PLICing (phosphorothioate-based ligase-independent gene cloning). The promoter toolbox was firstly validated using three well producible proteins (a cellulase from a metagenome library, a phytase from Yersinia mollaretii and an alcohol dehydrogenase from Pseudomonas putida) and then applied to two enzymes (3D1 DNA polymerase and glutamate dehydrogenase mutant) which are poorly produced in E. coli. By applying our PLICable pET-promoter toolbox, the authors were able to increase production by two-fold for 3D1 DNA polymerase (lac promoter) and 29-fold for glutamate dehydrogenase mutant H52Y (trc promoter).

RNA polymerase supply and flux through the lac operon in Escherichia coli.

Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli. By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase.This article is part of the themed issue ‘The new bacteriology’.

Protein-induced DNA linking number change by sequence-specific DNA binding proteins and its biological effects.

Sequence-specific DNA-binding proteins play essential roles in many fundamental biological events such as DNA replication, recombination, and transcription. One common feature of sequence-specific DNA-binding proteins is to introduce structural changes to their DNA recognition sites including DNA-bending and DNA linking number change (ΔLk). In this article, I review recent progress in studying protein-induced ΔLk by several sequence-specific DNA-binding proteins, such as E. coli cAMP receptor protein (CRP) and lactose repressor (LacI). It was demonstrated recently that protein-induced ΔLk is an intrinsic property for sequence-specific DNA-binding proteins and does not correlate to protein-induced other structural changes, such as DNA bending. For instance, although CRP bends its DNA recognition site by 90°, it was not able to introduce a ΔLk to it. However, LacI was able to simultaneously bend and introduce a ΔLk to its DNA binding sites. Intriguingly, LacI also constrained superhelicity within LacI-lac O1 complexes if (-) supercoiled DNA templates were provided. I also discuss how protein-induced ΔLk help sequence-specific DNA-binding proteins regulate their biological functions. For example, it was shown recently that LacI utilizes the constrained superhelicity (ΔLk) in LacI-lac O1 complexes and serves as a topological barrier to constrain free, unconstrained (-) supercoils within the 401-bp DNA loop. These constrained (-) supercoils enhance LacI's binding affinity and therefore the repression of the lac promoter. Other biological functions include how DNA replication initiators λ O and DnaA use the induced ΔLk to open/melt bacterial DNA replication origins.

Supercoiling Effects on Short-Range DNA Looping in E. coli

DNA-protein loops can be essential for gene regulation. The Escherichia coli lactose (lac) operon is controlled by DNA-protein loops that have been studied for decades. Here we adapt this model to test the hypothesis that negative superhelical strain facilitates the formation of short-range (6–8 DNA turns) repression loops in E. coli. The natural negative superhelicity of E. coli DNA is regulated by the interplay of gyrase and topoisomerase enzymes, adding or removing negative supercoils, respectively. Here, we measured quantitatively DNA looping in three different E. coli strains characterized by different levels of global supercoiling: wild type, gyrase mutant (gyrB226), and topoisomerase mutant (ΔtopA10). DNA looping in each strain was measured by assaying repression of the endogenous lac operon, and repression of ten reporter constructs with DNA loop sizes between 70–85 base pairs. Our data are most simply interpreted as supporting the hypothesis that negative supercoiling facilitates gene repression by small DNA-protein loops in living bacteria.

Construction, properties, and application of the pCB5 plasmid, a novel conjugative shuttle vector with a Cupriavidus basilensis origin of replication.

Cupriavidus basilensis is a species with diverse metabolic capabilities, including degradation of xenobiotics and heavy metal resistance. Although the genomes of several strains of this species have been sequenced, no plasmid has yet been constructed for genetic engineering in this species. In this study, we identified a novel plasmid, designated pWS, from C. basilensis WS with a copy number of 1-3 per cell and a length of 2150bp. pWS contained three protein-coding genes, among which only rep was required for plasmid replication. Rep showed no homology with known plasmid replication initiators. Unlike most plasmids, pWS did not have a cis-acting replication origin outside the region of rep. The minimal replicon of pWS was stable in C. basilensis WS without selection. A conjugative C. basilensis/Escherichia coli shuttle vector, pCB5, was constructed using the minimal replicon of pWS. Interestingly, the copy number of pCB5 was flexible and could be manipulated. Enhancing the expression level of Rep in pCB5 by either doubling the promoter or coding region of rep resulted in doubling of the plasmid copy number. Moreover, replacing the native promoter of rep with the lac promoter increased the copy number by over fivefold. Finally, using two different β-galactosidase reporting systems constructed with pCB5, we successfully demonstrated the different regulatory patterns of bph and dmp operons during diphenyl ether (DE) degradation in C. basilensis WS. Thus, this shuttle vector provided an efficient tool for DNA cloning and metabolic engineering in C. basilensis.

Comparative genomics of Clostridium bolteae and Clostridium clostridioforme reveals species-specific genomic properties and numerous putative antibiotic resistance determinants.

Background Clostridium bolteae and Clostridium clostridioforme, previously included in the complex C. clostridioforme in the group Clostridium XIVa, remain difficult to distinguish by phenotypic methods. These bacteria, prevailing in the human intestinal microbiota, are opportunistic pathogens with various drug susceptibility patterns. In order to better characterize the two species and to obtain information on their antibiotic resistance genes, we analyzed the genomes of six strains of C. bolteae and six strains of C. clostridioforme, isolated from human infection.ResultsThe genome length of C. bolteae varied from 6159 to 6398 kb, and 5719 to 6059 CDSs were detected. The genomes of C. clostridioforme were smaller, between 5467 and 5927 kb, and contained 5231 to 5916 CDSs. The two species display different metabolic pathways. The genomes of C. bolteae contained lactose operons involving PTS system and complex regulation, which contribute to phenotypic differentiation from C. clostridioforme. The Acetyl-CoA pathway, similar to that of Faecalibacterium prausnitzii, a major butyrate producer in the human gut, was only found in C. clostridioforme. The two species have also developed diverse flagella mobility systems contributing to gut colonization. Their genomes harboured many CDSs involved in resistance to beta-lactams, glycopeptides, macrolides, chloramphenicol, lincosamides, rifampin, linezolid, bacitracin, aminoglycosides and tetracyclines. Overall antimicrobial resistance genes were similar within a species, but strain-specific resistance genes were found. We discovered a new group of genes coding for rifampin resistance in C. bolteae. C. bolteae 90B3 was resistant to phenicols and linezolide in producing a 23S rRNA methyltransferase. C. clostridioforme 90A8 contained the VanB-type Tn1549 operon conferring vancomycin resistance. We also detected numerous genes encoding proteins related to efflux pump systems.ConclusionGenomic comparison of C. bolteae and C. clostridiofrome revealed functional differences in butyrate pathways and in flagellar systems, which play a critical role within human microbiota. Most of the resistance genes detected in both species were previously characterized in other bacterial species. A few of them were related to antibiotics inactive against Clostridium spp. Some were part of mobile genetic elements suggesting that these commensals of the human microbiota act as reservoir of antimicrobial resistances.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3152-x) contains supplementary material, which is available to authorized users.

Affinity purification of bacterial outer membrane vesicles (OMVs) utilizing a His-tag mutant

To facilitate the rapid purification of bacterial outer membrane vesicles (OMVs), we developed two plasmid constructs that utilize a truncated, transmembrane protein to present an exterior histidine repeat sequence. We chose OmpA, a highly abundant porin protein, as the protein scaffold and utilized the lac promoter to allow for inducible control of the epitope-presenting construct. OMVs containing mutant OmpA-His6 were purified directly from Escherichia coli culture media on an immobilized metal affinity chromatography (IMAC) Ni-NTA resin. This enabling technology can be combined with other molecular tools directed at OMV packaging to facilitate the separation of modified/cargo-loaded OMV from their wt counterparts. In addition to numerous applications in the pharmaceutical and environmental remediation industries, this technology can be utilized to enhance basic research capabilities in the area of elucidating endogenous OMV function.

Sortase A-Mediated Metabolic Enzyme Ligation in Escherichia coli.

We demonstrate metabolic enzyme ligation using a transpeptidase (Staphylococcal sortase A) in the microbial cytoplasm for the redirection of metabolic flux through metabolic channeling. Here, sortase A expression was controlled by the lac promoter to trigger metabolic channeling by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG). We tested covalent linking of pyruvate-formate lyase and phosphate acetyltransferase by sortase A-mediated ligation and evaluated the production of acetate. The time point of addition of IPTG was not critical for facilitating metabolic enzyme ligation, and acetate production increased upon expression of sortase A. These results show that sortase A-mediated enzyme ligation enhances an acetate-producing flux in E. coli. We have validated that sortase A-mediated enzyme ligation offers a metabolic channeling approach to redirect a central flux to a desired flux.

A self-inducible heterologous protein expression system in Escherichia coli.

Escherichia coli is an important experimental, medical and industrial cell factory for recombinant protein production. The inducible lac promoter is one of the most commonly used promoters for heterologous protein expression in E. coli. Isopropyl-β-D-thiogalactoside (IPTG) is currently the most efficient molecular inducer for regulating this promoter’s transcriptional activity. However, limitations have been observed in large-scale and microplate production, including toxicity, cost and culture monitoring. Here, we report the novel SILEX (Self-InducibLe Expression) system, which is a convenient, cost-effective alternative that does not require cell density monitoring or IPTG induction. We demonstrate the broad utility of the presented self-inducible method for a panel of diverse proteins produced in large amounts. The SILEX system is compatible with all classical culture media and growth temperatures and allows protein expression modulation. Importantly, the SILEX system is proven to be efficient for protein expression screening on a microplate scale.

Ensifer meliloti overexpressing Escherichia coli phytase gene (appA) improves phosphorus (P) acquisition in maize plants.

The Escherichia coli phytase gene appA encoding enzyme AppA was cloned in a broad host range plasmid pBBR1MCS2 (lac promoter), termed pVA1, and transformed into the Ensifer meliloti 1020. Transformation of pVA1 in Ensifer meliloti {E. m (pVA1)} increased its phosphatase and phytase activity by ∼9- and ∼50-fold, respectively, compared to the transformants containing empty plasmid as control {E. m (pBBR1MCS2)}. The western blot experiments using rabbit anti-AppA antibody showed that AppA is translocated into the periplasm of the host after its expression. Ensifer meliloti harboring AppA protein {E. m (pVA1)} and {E. m (pBBR1MCS2)} could acidify the unbuffered phytate minimal media (pH 8.0) containing Ca-phytate or Na-phytate as sole organic P (Po) source to below pH 5.0 and released P. However, both {E. m (pVA1)} and {E. m (pBBR1MCS2)} neither dropped pH of the medium nor released P when the medium was buffered at pH 8.0 using Tris-Cl, indicating that acidification of medium was important for the enzymatic hydrolysis of phytate. Further experiments proved that maize plants inoculated with {E. m. (pVA1)} showed increase in growth under sterile semi solid agar (SSA) medium containing Na-phytate as sole P source. The present study could be helpful in generating better transgenic bioinoculants harboring phosphate mineralization properties that ultimately promote plant growth.

RNA Polymerase Pausing during Initial Transcription

SummaryIn bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause (“initiation pause,” ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.

Construction of chromosomally encoded lacZ and gfp reporter strains of Escherichia coli for the study of global regulation of metabolism

We developed plasmid systems pJLIC and pJLRCS that allow the easy cloning of promoter regions upstream of reporter genes (lacZ and gfp) and the subsequent transfer of the resulting reporter cassettes into the chromosome of Escherichia coli by recombineering. Thus, a promoter region can be inserted upstream of lacZ gene in the context of the lactose operon of recipient E. coli strains. Moreover, a promoter–GFP reporter is inserted at a nonessential site of the chromosome. These systems allow for selection and promoter strength analysis at the single copy level. We also provide a collection of E. coli knock‐out mutants in genes for global regulation of C, N, and O metabolism, and combinations of these mutants with the newly constructed chromosomal lacZ and gfp reporter system. This allows assaying transcriptional activities of promoters before the background of different mutants. Both lacZ and gfp reporter cassettes (including appropriate antibiotic resistance markers) and the regulator knock‐outs may be easily transferred by phage P1 mediated transduction to other E. coli strains. As a proof of principle, ß‐galactosidase activity and GFP fluorescence were compared for lacZ and gfp reporter systems placed under the control of the PrssA promoter both in a wild‐type and an isogenic rpoS‐mutant strain.

Production of steviol from steviol glucosides using β-glycosidase from Sulfolobus solfataricus.

Steviol is a diterpene isolated from the plant Stevia rebaudiana that has a potential role as an antihyperglycemic agent by stimulating insulin secretion from pancreatic beta cells and also has significant potential to diminish the renal clearance of anionic drugs and their metabolites. In this study, the lacS gene, which encodes a thermostable β-glycosidase (SSbgly) enzyme from the extremely thermoacidophillic archaeon Sulfolobus solfataricus, was cloned and expressed in E. coli Rossetta BL21(DE3)pLyS using lactose as an inducer. Through fermentation, SSbgly was expressed as a 61kDa protein with activity of 24.3U/mg and the OD600 of 23 was reached after 18h induction with 10mM lactose. Purified protein was obtained by Ni-Sepharose chromatography with a yield of 92.3%. SSbgly hydrolyzed steviol glycosides to produce steviol with a yield of 99.2%. The optimum conditions for steviol production were 50U/ml SSbgly and 90mg/ml Ste at 75°C as determined by the response surface method.

Crustal contamination and sulfide immiscibility history of the Permian Huangshannan magmatic Ni-Cu sulfide deposit, East Tianshan, NW China

The Huangshannan mafic–ultramafic intrusion is a Permian Ni–Cu sulfide-bearing intrusion in the southern margin of the Central Asian Orogenic Belt. The intrusion consists of an ultramafic unit, which is composed of lherzolite and olivine websterite, and a mafic unit, which is composed of olivine gabbronorite, gabbronorite and leuco-gabbronorite. This intrusion was formed by two separate pulses of magma: a more primitive magma for the early ultramafic unit and a more evolved magma for the late mafic unit. U–Pb isotope geochronology of zircon from the mafic unit yields an age of 278±2Ma. According to its olivine and Cr-rich spinel compositions, the estimated parental magma of lherzolite for the Huangshannan intrusion has 12.4wt.% MgO, indicating picritic affinity. Fractional crystallization modeling results and the presence of rounded sulfide inclusions in an olivine crystal (Fo 86.7) indicate that sulfide immiscibility was achieved at the beginning of olivine fractionation. Co-magmatic zircon crystals from gabbronorite have a δ18O value close to 6.5‰, which is 1.2‰ higher than the typical mantle value and suggests significant crustal contamination (∼20%). The positive εHf(t) values of co-magmatic zircon (which vary from +9.2 to +15.3) and positive whole rock εNd(t) values (which vary from +4.7 to +7.8) also indicate that the parental magma was derived from a depleted mantle source and contaminated by 5–20% juvenile arc crust and then by ∼5% upper crustal materials. However, modeling results of sulfur content at sulfide saturation reveal that such a large amount of crustal contamination is not sufficient to trigger sulfide saturation in the parental magma, which strongly suggests that external sulfur addition, probably during contamination, has played a critical role in causing sulfide immiscibility. Furthermore, the arc magmatism geochemical signatures of the Huangshannan intrusion, such as significant Nb and Ta depletion relative to La and low Ca contents in olivine, are interpreted as the result of slab-derived fluid modification during previous subduction. We speculate that the contamination of juvenile arc crust may have occurred in the lower crust because of picritic magma underplating and that partial melting in the mantle was triggered by the impingement of a mantle plume or lithosphere delamination in a post-subduction environment.

Boolean network representation of a continuous-time system and finite-horizon optimal control: application to the single-gene regulatory system for the lac operon

ABSTRACTIn this paper, the single-gene regulatory system for the lac operon is considered and a method to build a Boolean network able to represent its dynamics is given. A procedure, based on techniques borrowed from algebraic geometry, to compute an optimal control policy minimising a cost function is given for such a Boolean network. A tool to apply such an optimal control policy to the continuous-time system is reported. The proposed control method is tested in two simulative examples.

Improved Determination of Subnuclear Position Enabled by Three-Dimensional Membrane Reconstruction

Many aspects of chromatin biology are influenced by the nuclear compartment in which a locus resides, from transcriptional regulation to DNA repair. Further, the dynamic and variable localization of a particular locus across cell populations and over time makes analysis of a large number of cells critical. As a consequence, robust and automatable methods to measure the position of individual loci within the nuclear volume in populations of cells are necessary to support quantitative analysis of nuclear position. Here, we describe a three-dimensional membrane reconstruction approach that uses fluorescently tagged nuclear envelope or endoplasmic reticulum membrane marker proteins to precisely map the nuclear volume. This approach is robust to a variety of nuclear shapes, providing greater biological accuracy than alternative methods that enforce nuclear circularity, while also describing nuclear position in all three dimensions. By combining this method with established approaches to reconstruct the position of diffraction-limited chromatin markers—in this case, lac Operator arrays bound by lacI-GFP—the distribution of loci positions within the nuclear volume with respect to the nuclear periphery can be quantitatively obtained. This stand-alone image analysis pipeline should be of broad practical utility for individuals interested in various aspects of chromatin biology, while also providing, to our knowledge, a new conceptual framework for investigators who study organelle shape.