Abstract:
:Gene function is typically evaluated by sampling the continuum of gene expression at only a few discrete points corresponding to gene knockout or overexpression. We argue that this characterization is incomplete and present a library of engineered promoters of varying strengths obtained through mutagenesis of a constitutive promoter. A multifaceted characterization of the library, especially at the single-cell level to ensure homogeneity, permitted quantitative assessment correlating the effect of gene expression levels to improved growth and product formation phenotypes in Escherichia coli. Integration of these promoters into the chromosome can allow for a quantitative accurate assessment of genetic control. To this end, we used the characterized library of promoters to assess the impact of phosphoenolpyruvate carboxylase levels on growth yield and deoxy-xylulose-P synthase levels on lycopene production. The multifaceted characterization of promoter strength enabled identification of optimal expression levels for ppc and dxs, which maximized the desired phenotype. Additionally, in a strain preengineered to produce lycopene, the response to deoxy-xylulose-P synthase levels was linear at all levels tested, indicative of a rate-limiting step, unlike the parental strain, which exhibited an optimum expression level, illustrating that optimal gene expression levels are variable and dependent on the genetic background of the strain. This promoter library concept is illustrated as being generalizable to eukaryotic organisms (Saccharomyces cerevisiae) and thus constitutes an integral platform for functional genomics, synthetic biology, and metabolic engineering endeavors.
journal_name
Proc Natl Acad Sci U S Aauthors
Alper H,Fischer C,Nevoigt E,Stephanopoulos Gdoi
10.1073/pnas.0504604102keywords:
subject
Has Abstractpub_date
2005-09-06 00:00:00pages
12678-83issue
36eissn
0027-8424issn
1091-6490pii
0504604102journal_volume
102pub_type
杂志文章abstract::The method of matrix isolation has been used to investigate mechanisms of gas-phase chemical reactions, in particular the pyrolysis of some organomercury compounds. A molecular beam of pyrolysis products was condensed simultaneously with a large excess of rare gas at temperatures from 5 to 15 degrees K to form a matri...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.68.12.3238
更新日期:1971-12-01 00:00:00
abstract::Peptidoglycan (PGN) recognition proteins (PGRPs) are pattern-recognition receptors of the innate immune system that bind and, in some cases, hydrolyze bacterial PGNs. We determined the crystal structure, at 2.30-A resolution, of the C-terminal PGN-binding domain of human PGRP-Ialpha in complex with a muramyl tripeptid...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0407856101
更新日期:2004-12-07 00:00:00
abstract::Many processes in science and engineering develop multiscale temporal and spatial patterns, with complex underlying dynamics and time-dependent external forcings. Because of the importance in understanding and predicting these phenomena, extracting the salient modes of variability empirically from incomplete observati...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1118984109
更新日期:2012-02-14 00:00:00
abstract::Profilin2 (PFN2) is a target of the embryonic stem cell (ESC)-enriched miR-290 family of microRNAs (miRNAs) and an actin/dynamin-binding protein implicated in endocytosis. Here we show that the miR-290-PFN2 pathway regulates many aspects of ESC biology. In the absence of miRNAs, PFN2 is up-regulated in ESCs, with a re...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.2002750117
更新日期:2020-08-25 00:00:00
abstract::In the traditional Z scheme of photosynthesis the Emerson effects of red drop (decline in yield of photosynthesis in far-red light) and enhancement (of far-red yield by supplementary short-wavelength light) are taken to be evidence for the coupling in series of two photosystems that absorb unsymmetrically in the far-r...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.84.12.4103
更新日期:1987-06-01 00:00:00
abstract::The phenotype of several erythromycin-resistant mutants of Chlamydomonas reinhardi was further characterized in terms of the electrophoretic properties of their chloroplast ribosomal proteins. In mutant ery-M2d a single protein of the large (52 S) subunit has altered properties, which probably result from a change in ...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.69.12.3779
更新日期:1972-12-01 00:00:00
abstract::A type of heritable cellular change, known as habituation, occurs spontaneously in plant tissue and cell culture. This phenomenon is characterized by a newly acquired capacity of plant cells to produce growth regulatory substances. Using cloned lines of tobacco pith parenchyma cells, we demonstrated that a newly acqui...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.70.9.2660
更新日期:1973-09-01 00:00:00
abstract::Theoretical considerations predict that amplification of expressed gene transcripts by reverse transcription-PCR using arbitrarily chosen primers will result in the preferential amplification of the central portion of the transcript. Systematic, high-throughput sequencing of such products would result in an expressed ...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.97.7.3491
更新日期:2000-03-28 00:00:00
abstract::Executing gene circuits by cell-free transcription-translation into cell-sized compartments, such as liposomes, is one of the major bottom-up approaches to building minimal cells. The dynamic synthesis and proper self-assembly of macromolecular structures inside liposomes, the cytoskeleton in particular, stands as a c...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1914656117
更新日期:2020-01-28 00:00:00
abstract::The chromophore of photoactive yellow protein (PYP) (i.e., 4-hydroxycinnamic acid) has been replaced by an analogue with a triple bond, rather than a double bond (by using 4-hydroxyphenylpropiolic acid in the reconstitution, yielding hybrid I) and by a "locked" chromophore (through reconstitution with 7-hydroxycoumari...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.95.13.7396
更新日期:1998-06-23 00:00:00
abstract::Amoebae of the cellular slime mold Dictyostelium discoideum possess an extensive and dynamic endomembrane system that includes many types of acidic vacuoles. A light membrane fraction from Dictyostelium, rich in vacuolar-type H(+)-ATPase, has been described [Padh, H., Lavasa, M. & Steck, T.L. (1989) J. Cell Biol. 108,...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.89.17.8025
更新日期:1992-09-01 00:00:00
abstract::We have earlier proposed a molecular mechanism for the translocation of hydrophilic proteins across membranes that accounts for the experimental facts and meets the restrictions that we stipulate for such a mechanism. In particular, the restrictions are that translocation occurs by successive segments of the polypepti...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.84.7.1960
更新日期:1987-04-01 00:00:00
abstract::Mosquitoes are major disease vectors because most species must feed on blood from a vertebrate host to produce eggs. Blood feeding by the vector mosquito Aedes aegypti triggers the release of two neurohormones, ovary ecdysteroidogenic hormone (OEH) and insulin-like peptides (ILPs), which activate multiple processes re...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1501814112
更新日期:2015-04-21 00:00:00
abstract::Mass cytometry enables an unprecedented number of parameters to be measured in individual cells at a high throughput, but the large dimensionality of the resulting data severely limits approaches relying on manual "gating." Clustering cells based on phenotypic similarity comes at a loss of single-cell resolution and o...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1321405111
更新日期:2014-01-07 00:00:00
abstract::The orphan nuclear receptor Nur77/N10 has recently been demonstrated to be involved in apoptosis of T cell hybridomas. We report here that chronic expression of Nur77/N10 in thymocytes of transgenic mice results in a dramatic reduction of CD4+CD8+ double-positive as well as CD4+CD8- and CD4-CD8+ single-positive cell p...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.93.11.5533
更新日期:1996-05-28 00:00:00
abstract::Solvent additives (cosolvents, osmolytes) modulate biochemical reactions if, during the course of the reaction, there is a change in preferential interactions of solvent components with the reacting system. Preferential interactions can be expressed in terms of preferential binding of the cosolvent or its preferential...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.122225399
更新日期:2002-07-23 00:00:00
abstract::Ascorbate (vitamin C) recycling occurs when extracellular ascorbate is oxidized, transported as dehydroascorbic acid, and reduced intracellularly to ascorbate. We investigated microorganism induction of ascorbate recycling in human neutrophils and in microorganisms themselves. Ascorbate recycling was determined by mea...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.94.25.13816
更新日期:1997-12-09 00:00:00
abstract::The knowledge of tropical palaeoclimates is crucial for understanding global climate change, because it is a test bench for general circulation models that are ultimately used to predict future global warming. A longstanding issue concerning the last glacial maximum in the tropics is the discrepancy between the decrea...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0610109104
更新日期:2007-06-05 00:00:00
abstract::Antarctic ozone depletion is associated with enhanced chlorine from anthropogenic chlorofluorocarbons and heterogeneous chemistry under cold conditions. The deep Antarctic "hole" contrasts with the generally weaker depletions observed in the warmer Arctic. An unusually cold Arctic stratospheric season occurred in 2011...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1319307111
更新日期:2014-04-29 00:00:00
abstract::Current issues pertaining to theoretical simulations of materials, with a focus on systems of nanometer-scale dimensions, are discussed. The use of atomistic simulations as high-resolution numerical experiments, enabling and guiding formulation and testing of analytic theoretical descriptions, is demonstrated through ...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0408038102
更新日期:2005-05-10 00:00:00
abstract::Y chromosomes control essential male functions in many species, including sex determination and fertility. However, because of obstacles posed by repeat-rich heterochromatin, knowledge of Y chromosome sequences is limited to a handful of model organisms, constraining our understanding of Y biology across the tree of l...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1525164113
更新日期:2016-04-12 00:00:00
abstract::Growth inhibition assays indicated that the IC50 values for methotrexate (MTX) and 5-fluorodeoxyuridine (FdUrd) in HS-18, a liposarcoma cell line lacking retinoblastoma protein (pRB), and SaOS-2, an osteosarcoma cell line with a truncated and nonfunctional pRB, were 10- to 12-fold and 4- to 11-fold higher, respectivel...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.92.22.10436
更新日期:1995-10-24 00:00:00
abstract::Fluorescence in situ hybridization (FISH) resolution has advanced because newer techniques use increasingly decondensed chromatin. FISH cannot analyze restriction enzyme cutting sites due to limitations of the hybridization and detection technologies. The RecA-assisted restriction endonuclease (RARE) technique cleaves...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.92.1.165
更新日期:1995-01-03 00:00:00
abstract::C1q, a member of the immune complement cascade, is implicated in the selective pruning of synapses by microglial phagocytosis. C1q-mediated synapse elimination has been shown to occur during brain development, while increased activation and complement-dependent synapse loss is observed in neurodegenerative diseases. H...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1722613115
更新日期:2018-06-12 00:00:00
abstract::During the entry process, the human papillomavirus (HPV) capsid is trafficked to the trans-Golgi network (TGN), whereupon it enters the nucleus during mitosis. We previously demonstrated that the minor capsid protein L2 assumes a transmembranous conformation in the TGN. Here we provide evidence that the incoming viral...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1600638113
更新日期:2016-05-31 00:00:00
abstract::We have shown previously that guanine nucleotide-binding protein (G protein) beta gamma complexes purified from bovine brain membranes are methyl esterified on a C-terminal cysteine residue of the gamma polypeptide. In the present study, 3H-methylated G beta gamma complexes cleaved to their constituent amino acids by ...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.87.15.5868
更新日期:1990-08-01 00:00:00
abstract::Insect damage on fossil leaves from the Central Rocky Mountains, United States, documents the response of herbivores to changing regional climates and vegetation during the late Paleocene (humid, warm temperate to subtropical, predominantly deciduous), early Eocene (humid subtropical, mixed deciduous and evergreen), a...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.111069498
更新日期:2001-05-22 00:00:00
abstract::We used a modified subtractive suppression hybridization to identify cellular genes that show altered expression in Burkitt lymphomas (BLs) in the presence of Epstein-Barr virus (EBV). Comparison of the gene expression patterns of an EBV-negative clone of the originally EBV-positive BL line Akata, with its Neo(R)-EBV ...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0730710100
更新日期:2003-04-15 00:00:00
abstract::Targeting neuroendocrine tumors expressing somatostatin receptor subtypes (sst) with radiolabeled somatostatin agonists is an established diagnostic and therapeutic approach in oncology. While agonists readily internalize into tumor cells, permitting accumulation of radioactivity, radiolabeled antagonists do not, and ...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0607761103
更新日期:2006-10-31 00:00:00
abstract::Complexin activates Ca(2+)-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca(2+)-sensor synaptotagmin. The N-terminal domain of complexin is importa...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1604348113
更新日期:2016-08-09 00:00:00