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Experimental and Theoretical Considerations of P1-plasmid Replication and Segregation During the E. coli Cell Cycle
Stephen Cooper,Jay D. Keasling
Journal of Biological Sciences , 2005,
Abstract: Two contrasting experimental descriptions of P1-plasmid replication during the cell cycle of Escherichia coli have been described. One set of results led to the proposal that replication of P1-plasmid occurs at a specific time during the cell cycle over a wide range of growth rates and follows rules similar to that governing bacterial chromosome replication. Experiments supporting this proposal utilized membrane-elution experiments, radioactive double-labeling of DNA and scintillation counting of purified plasmids. An alternative experimental description of P1-plasmid replication during the cell cycle, also based on membrane-elution methodology but measuring radioactivity incorporated into plasmid DNA by autoradiography and scanning of films, proposed that P1-plasmid replicates at all stages of the cell cycle in rapidly growing cells, but with a slight periodicity or increase in P1 replication probability within the cell cycle of slower growing cells. These discordant experimental results are analyzed. It is concluded that the direct double-label counting approach is to be preferred, as the results are consistent with a large number of experiments, are supported by theoretical considerations and yield a unified view of plasmid replication over a wide range of growth rates. Theoretical ramifications of each view of P1-plasmid replication-cycle-dependent and cycle-independent are compared. An analysis of P1-plasmid segregation is also presented.
Kinetics of Phosphomevalonate Kinase from Saccharomyces cerevisiae
David E. Garcia, Jay D. Keasling
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0087112
Abstract: The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in Escherichia coli has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC from Saccharomyces cerevisiae, a previously unstudied enzyme. An E. coli codon-optimized version of the S. cerevisiae gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in E. coli BL21(DE3) and purified on a Ni2+ column. The KM of the ATP binding site was determined to be 98.3 μM at 30°C, the optimal growth temperature for S. cerevisiae, and 74.3 μM at 37°C, the optimal growth temperature for E. coli. The KM of the mevalonate-5-phosphate binding site was determined to be 885 μM at 30°C and 880 μM at 37°C. The Vmax was determined to be 4.51 μmol/min/mg enzyme at 30°C and 5.33 μmol/min/mg enzyme at 37°C. PMK is Mg2+ dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH = 7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 μM FPP.
A rapid and inexpensive labeling method for microarray gene expression analysis
Mario Ouellet, Paul D Adams, Jay D Keasling, Aindrila Mukhopadhyay
BMC Biotechnology , 2009, DOI: 10.1186/1472-6750-9-97
Abstract: Two total RNA samples were labeled with each method and hybridized to NimbleGen expression arrays. Although all methods tested here provided similar global results and biological conclusions, the new direct random-primed cDNA labeling method provided slightly better correlation between replicates compared to the other methods and thus increased ability to find statistically significant differentially expressed genes.The new direct random-primed cDNA labeling method introduced here is suitable for gene expression microarrays and provides a rapid, inexpensive alternative to existing methods. Using NimbleGen microarrays, the method produced excellent results comparable to those obtained with other methods. However, the simplicity and cost-effectiveness of the new method allows for increased sample throughput in microarray experiments and makes the process amenable to automation with a relatively simple liquid handling system.DNA microarrays allow global profiling of nucleic acid sequences and have become an important and ubiquitous tool in biological and biomedical research. Although many applications of DNA microarrays have been developed in the past decade [1,2], differential gene expression profiling remains the most widely used application of this technology. Improvements in microarray design now allow rapid fabrication of custom microarrays, representation of an increasingly large number of features on a single glass slide and hybridization of multiple samples on physically separated arrays on the same slide. Robots designed specifically for DNA and RNA extraction are also commercially available now and can considerably reduce the hands-on time required for RNA preparation for microarray studies. Although identification of the most biologically relevant information from a microarray experiment and interpretation of this information in a biological context can be challenging, methods and tools for microarray data analysis have become more widely available and easy
A framework and model system to investigate linear system behavior in Escherichia coli
Meghdad Hajimorad, Paul R Gray, Jay D Keasling
Journal of Biological Engineering , 2011, DOI: 10.1186/1754-1611-5-3
Abstract: We developed a framework and model system consisting of three devices to investigate linear system behavior in E. coli. Our framework employed the transfer curve concept to determine the amount of nonlinearity elicited by the E. coli transcriptional system in response to the devices. To this effect, the model system was quantitatively characterized using real-time quantitative PCR to produce device transfer curves (DTCs). Two of the devices encoded the bacterial neomycin phosphotransferase II (nptII) and chloramphenicol acetyl transferase (cat), while the third encoded the jellyfish-originating green fluorescent protein (gfp). The gfp device was the most nonlinear in our system, with nptII and cat devices eliciting linear responses. Superposition experiments verified these findings, with independence among the three devices having been lost when gfp was present at copy numbers above the lowest one used.We show that linear system behavior is possible in E. coli. Elucidation of the mechanism underlying the nonlinearity observed in gfp may lead to design rules that ensure linear system behavior, enabling the accurate prediction of the quantitative behavior of a system assembled from individually characterized devices. Our work suggests that biological systems follow principles similar to physical ones, and that concepts borrowed from the latter (such as DTCs) may be of use in the characterization and design of biological systems.Engineering biological systems with predictable, quantitative behavior is currently a challenging problem. Presently, this requires months (at times years) of trial-and-error type of experiments, with the engineering of functional systems being more akin to art than engineering [1]. Synthetic biology aims to develop foundational principles and technologies that will enable the systematic forward engineering of biological systems [2-4]. In particular, synthetic biology aims to develop frameworks that apply the engineering principles of abstracti
Functional Characterization of the Origin of Replication of pRN1 from Sulfolobus islandicus REN1H1
Chijioke J. Joshua, Luis D. Perez, Jay D. Keasling
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0084664
Abstract: Plasmid pRN1 from Sulfolobus islandicus REN1H1 is believed to replicate by a rolling circle mechanism but its origin and mechanism of replication are not well understood. We sought to create minimal expression vectors based on pRN1 that would be useful for heterologous gene expression in S. acidocaldarius, and in the process improve our understanding of the mechanism of replication. We constructed and transformed shuttle vectors that harbored different contiguous stretches of DNA from pRN1 into S. acidocaldarius E4-39, a uracil auxotroph. A 232-bp region 3’ of orf904 was found to be critical for pRN1 replication and is therefore proposed to be the putative origin of replication. This 232-bp region contains a 100-bp stem-loop structure believed to be the double-strand origin of replication. The loop of the 100-bp structure contains a GTG tri-nucleotide motif, a feature that was previously reported to be important for the primase activity of Orf904. This putative origin and the associated orf56 and orf904 were identified as the minimal replicon of pRN1 because transformants of plasmids lacking any of these three features were not recovered. Plasmids lacking orf904 and orf56 but harboring the putative origin were transformable when orf904 and orf56 were provided in-trans; a 75-bp region 5’ of the orf904 start codon was found to be essential for this complementation. Detailed knowledge of the pRN1 origin of replication will broaden the application of the plasmid as a genetic tool for Sulfolobus species.
Design and Construction of a Double Inversion Recombination Switch for Heritable Sequential Genetic Memory
Timothy S. Ham, Sung K. Lee, Jay D. Keasling, Adam P. Arkin
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0002815
Abstract: Background Inversion recombination elements present unique opportunities for computing and information encoding in biological systems. They provide distinct binary states that are encoded into the DNA sequence itself, allowing us to overcome limitations posed by other biological memory or logic gate systems. Further, it is in theory possible to create complex sequential logics by careful positioning of recombinase recognition sites in the sequence. Methodology/Principal Findings In this work, we describe the design and synthesis of an inversion switch using the fim and hin inversion recombination systems to create a heritable sequential memory switch. We have integrated the two inversion systems in an overlapping manner, creating a switch that can have multiple states. The switch is capable of transitioning from state to state in a manner analogous to a finite state machine, while encoding the state information into DNA. This switch does not require protein expression to maintain its state, and “remembers” its state even upon cell death. We were able to demonstrate transition into three out of the five possible states showing the feasibility of such a switch. Conclusions/Significance We demonstrate that a heritable memory system that encodes its state into DNA is possible, and that inversion recombination system could be a starting point for more complex memory circuits. Although the circuit did not fully behave as expected, we showed that a multi-state, temporal memory is achievable.
Memory in Microbes: Quantifying History-Dependent Behavior in a Bacterium
Denise M. Wolf, Lisa Fontaine-Bodin, Ilka Bischofs, Gavin Price, Jay Keasling, Adam P. Arkin
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0001700
Abstract: Memory is usually associated with higher organisms rather than bacteria. However, evidence is mounting that many regulatory networks within bacteria are capable of complex dynamics and multi-stable behaviors that have been linked to memory in other systems. Moreover, it is recognized that bacteria that have experienced different environmental histories may respond differently to current conditions. These “memory” effects may be more than incidental to the regulatory mechanisms controlling acclimation or to the status of the metabolic stores. Rather, they may be regulated by the cell and confer fitness to the organism in the evolutionary game it participates in. Here, we propose that history-dependent behavior is a potentially important manifestation of memory, worth classifying and quantifying. To this end, we develop an information-theory based conceptual framework for measuring both the persistence of memory in microbes and the amount of information about the past encoded in history-dependent dynamics. This method produces a phenomenological measure of cellular memory without regard to the specific cellular mechanisms encoding it. We then apply this framework to a strain of Bacillus subtilis engineered to report on commitment to sporulation and degradative enzyme (AprE) synthesis and estimate the capacity of these systems and growth dynamics to ‘remember’ 10 distinct cell histories prior to application of a common stressor. The analysis suggests that B. subtilis remembers, both in short and long term, aspects of its cell history, and that this memory is distributed differently among the observables. While this study does not examine the mechanistic bases for memory, it presents a framework for quantifying memory in cellular behaviors and is thus a starting point for studying new questions about cellular regulation and evolutionary strategy.
DeviceEditor visual biological CAD canvas
Joanna Chen, Douglas Densmore, Timothy S Ham, Jay D Keasling, Nathan J Hillson
Journal of Biological Engineering , 2012, DOI: 10.1186/1754-1611-6-1
Abstract: We report the development and deployment of web-based bioCAD software, DeviceEditor, which provides a graphical design environment that mimics the intuitive visual whiteboard design process practiced in biological laboratories. The key innovations of DeviceEditor include visual combinatorial library design, direct integration with scar-less multi-part DNA assembly design automation, and a graphical user interface for the creation and modification of design specification rules. We demonstrate how biological designs are rendered on the DeviceEditor canvas, and we present effective visualizations of genetic component ordering and combinatorial variations within complex designs.DeviceEditor liberates researchers from DNA base-pair manipulation, and enables users to create successful prototypes using standardized, functional, and visual abstractions. Open and documented software interfaces support further integration of DeviceEditor with other bioCAD tools and software platforms. DeviceEditor saves researcher time and institutional resources through correct-by-construction design, the automation of tedious tasks, design reuse, and the minimization of DNA assembly costs.The development of bioCAD software is paramount to our future capacity to rapidly design increasingly complex biological systems for the predictable and reproducible production of biofuels and bio-based chemicals [1]. When considering a DNA construction task, researchers must choose from a rapidly expanding list of candidate gene orthologs and expression systems. BioCAD tools (reviewed in [2-4]) make it possible to automatically query parts repositories for putative design components [5] and model the performance of candidate component combinations [6-9]. These software tools can also address design workflow bottlenecks by providing canvases for abstractly visualizing and arranging genetic components [10] and automating the design and execution of the DNA assembly process [11,12] (reviewed in [13,14]).Howe
Global analysis of host response to induction of a latent bacteriophage
Robin E Osterhout, Israel A Figueroa, Jay D Keasling, Adam P Arkin
BMC Microbiology , 2007, DOI: 10.1186/1471-2180-7-82
Abstract: We observed a temporally coordinated program of phage gene expression, with distinct early, middle and late transcriptional classes. Our study confirmed known host-phage interactions of induction of the heat shock regulon, escape replication, and suppression of genes involved in cell division and initiation of replication. We identified 728 E. coli genes responsive to prophage induction, which included pleiotropic stress response pathways, the Arc and Cpx regulons, and global regulators crp and lrp. Several hundred genes involved in central metabolism, energy metabolism, translation and transport were down-regulated late in induction. Though statistically significant, most of the changes in these genes were mild, with only 140 genes showing greater than two-fold change.Overall, we observe that prophage induction has a surprisingly low impact on host physiology. This study provides the first global dynamic picture of how host processes respond to lambda phage induction.Bacteriophage lambda has been studied for over 50 years and has served as a model for understanding genetic networks, control and development. Lambda is a temperate phage capable of undergoing divergent developmental pathways: lysis and lysogeny. Lytic development is lethal to host Escherichia coli, resulting in amplification and release of progeny phage. In the lysogenic state the phage integrates into the host chromosome, where it can silence lytic promoters and replicate quiescently as a prophage. Induction of lysis from the lysogenic state can be triggered by agents that damage DNA or interfere with replication, such as mitomycin C and UV light. The gene regulatory network underlying the lambda lifecycle has been studied in exhaustive detail, yet the switch continues to reveal new levels of complex regulation [1]. The mechanistic details of this switch have been elegantly reviewed elsewhere [2].The lambda gene regulatory network is composed of both phage and host factors, many of which interact wit
BglBrick vectors and datasheets: A synthetic biology platform for gene expression
Taek Lee, Rachel A Krupa, Fuzhong Zhang, Meghdad Hajimorad, William J Holtz, Nilu Prasad, Sung Lee, Jay D Keasling
Journal of Biological Engineering , 2011, DOI: 10.1186/1754-1611-5-12
Abstract: Here, we report the development and characterization of a library of expression vectors compatible with the BglBrick standard (BBF RFC 21). We have designed and constructed 96 BglBrick-compatible plasmids with a combination of replication origins, antibiotic resistance genes, and inducible promoters. These plasmids were characterized over a range of inducer concentrations, in the presence of non-cognate inducer molecules, and with several growth media, and their characteristics were documented in a standard format datasheet. A three plasmid system was used to investigate the impact of multiple origins of replication on plasmid copy number.The standardized collection of vectors presented here allows the user to rapidly construct and test the expression of genes with various combinations of promoter strength, inducible expression system, copy number, and antibiotic resistance. The quantitative datasheets created for these vectors will increase the predictability of gene expression, especially when multiple plasmids and inducers are utilized.Metabolic engineering, the redirection of metabolic pathways using genetic manipulation, plays an important role in a wide range of biological research including drug production, bioremediation, and biofuel production [1-5]. Metabolic pathways that lead to important drugs or chemicals are often multi-step processes involving many enzymes. In addition, controlling and coordinating the activity of each enzyme to achieve the optimal production of the target product is extremely complicated [6-9]. To construct an entire metabolic pathway in a heterologous host, the genes encoding the pathway enzymes often have to be constructed on multiple plasmids. Furthermore, the expression of each enzyme needs to be tuned to balance it with that of the other enzymes in the pathway and to reduce the metabolic burden on the host cell [6,9-11]. Recently, several advanced cloning methods using homologous recombination, such as Sequence and Ligation-Ind
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