Protein production by auto-induction in high-density shaking cultures
TL;DR: Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amounts of lactose.
About: This article is published in Protein Expression and Purification.The article was published on 2005-05-01. It has received 5395 citations till now. The article focuses on the topics: Lactose & Gene expression.
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TL;DR: A family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo are developed and provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.
Abstract: Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5-40-µm long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.
5,365 citations
TL;DR: A review of second generation biodiesel production systems using microalgae can be found in this paper, where the main advantages of second-generation microalgal systems are that they: (1) have a higher photon conversion efficiency (as evidenced by increased biomass yields per hectare): (2) can be harvested batch-wise nearly all-year-round, providing a reliable and continuous supply of oil: (3) can utilize salt and waste water streams, thereby greatly reducing freshwater use: (4) can couple CO2-neutral fuel production with CO2 sequestration: (
Abstract: The use of fossil fuels is now widely accepted as unsustainable due to depleting resources and the accumulation of greenhouse gases in the environment that have already exceeded the “dangerously high” threshold of 450 ppm CO2-e. To achieve environmental and economic sustainability, fuel production processes are required that are not only renewable, but also capable of sequestering atmospheric CO2. Currently, nearly all renewable energy sources (e.g. hydroelectric, solar, wind, tidal, geothermal) target the electricity market, while fuels make up a much larger share of the global energy demand (∼66%). Biofuels are therefore rapidly being developed. Second generation microalgal systems have the advantage that they can produce a wide range of feedstocks for the production of biodiesel, bioethanol, biomethane and biohydrogen. Biodiesel is currently produced from oil synthesized by conventional fuel crops that harvest the sun’s energy and store it as chemical energy. This presents a route for renewable and carbon-neutral fuel production. However, current supplies from oil crops and animal fats account for only approximately 0.3% of the current demand for transport fuels. Increasing biofuel production on arable land could have severe consequences for global food supply. In contrast, producing biodiesel from algae is widely regarded as one of the most efficient ways of generating biofuels and also appears to represent the only current renewable source of oil that could meet the global demand for transport fuels. The main advantages of second generation microalgal systems are that they: (1) Have a higher photon conversion efficiency (as evidenced by increased biomass yields per hectare): (2) Can be harvested batch-wise nearly all-year-round, providing a reliable and continuous supply of oil: (3) Can utilize salt and waste water streams, thereby greatly reducing freshwater use: (4) Can couple CO2-neutral fuel production with CO2 sequestration: (5) Produce non-toxic and highly biodegradable biofuels. Current limitations exist mainly in the harvesting process and in the supply of CO2 for high efficiency production. This review provides a brief overview of second generation biodiesel production systems using microalgae.
2,254 citations
TL;DR: The different approaches for the synthesis of recombinant proteins in E. coli are reviewed and recent progress in this ever-growing field is discussed.
Abstract: Escherichia coli is the organism of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of recombinant proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.
1,883 citations
Cites background from "Protein production by auto-inductio..."
...A tighter control can be achieved by the addition of 0.2–1% w/v glucose in the medium as rich media prepared with tryptone or peptone may contain the inducer lactose (Studier, 2005)....
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...In that report, the concept of autoinduction was developed (Studier, 2005)....
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...…poses a problem, the media can be buffered with phosphate salts at 50 mM. 2xYT, TB (Terrific Broth) and SB (Super Broth) media recipes are available elsewhere and have been shown to be superior to LB for reaching higher cell densities (Madurawe et al., 2000; Atlas, 2004; Studier, 2005)....
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...Not surprisingly, increasing the amount of peptone or yeast extract leads to higher cell densities (Studier, 2005)....
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Howard Hughes Medical Institute1, University of California, Davis2, Champalimaud Foundation3, University of Puerto Rico4, Medical Research Council5, Yale University6, Princeton University7, Rockefeller University8, Harvard University9, University of California, San Francisco10, Tata Institute of Fundamental Research11, University of California, Los Angeles12
TL;DR: GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3, which allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
Abstract: Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
1,179 citations
TL;DR: The computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination—a model reaction for proton transfer from carbon—with measured rate enhancements of up to 105 and multiple turnovers are described.
Abstract: The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination—a model reaction for proton transfer from carbon—with measured rate enhancements of up to 105 and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in kcat/Km (kcat/Km of 2,600 M-1s-1 and kcat/kuncat of >106). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future. The design of enzymes able to catalyse re-actions that are not catalysed by natural biocatalysts is a tremendous challenge for computational protein design. Rothlisberger et al. now report using computational protein design to generate eight novel enzymes able to catalyse the Kemp elimination — a model reaction for proton transfer from carbon. The activity of the designed enzymes was enhanced by directed in vitro evolution, thereby demonstrating a powerful strategy for the creation of novel enzymes. A computational protein design was used to generate eight enzymes that were able to catalyse the Kemp elimination, a model reaction for proton transfer from carbon. Directed evolution was used to enhance the catalytic activity of the designed enzymes, demonstrating that the combination of computational protein design and directed evolution is a highly effective strategy to create novel enzymes.
1,171 citations
References
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Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
215,169 citations
7,047 citations
"Protein production by auto-inductio..." refers background or methods in this paper
...Coding sequences for target proteins were cloned under control of the T7lac promoter and the upstream translation initiation signals of the T7 major capsid protein [2,4,11] by placing the initiation codon at the position of the NdeI site of pET-13a [12] or pET-24b (Novagen), or the NcoI site of pREX vectors (equivalent to the NcoI site of pET-11d [2]; to be described elsewhere), all of which confer resistance to kanamycin....
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...Such overgrowth can occur when expression vectors confer resistance to ampicillin, because secreted b-lactamase can degrade all of the ampicillin in the medium [1,2]....
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...Perhaps the ability to prevent induction is related to a need for higher levels of allolactose to induce expression from the T7lac promoter in a multi-copy plasmid, because higher than normal levels of lac repressor are present to ensure saturation of all of the repressor binding sites [2,4]....
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...An inducible T7 expression system is highly effective and widely used to produce RNAs and proteins from cloned coding sequences in the bacterium Escherichia coli [1,2]....
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...An effective means to reduce basal expression is to place the lac operator sequence (the binding site for lac repressor) just downstream of the start site of a T7 promoter, creating a T7lac promoter [2,4]....
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TL;DR: A gene expression system based on bacteriophage T7 RNA polymerase has been developed and high levels of accumulation suggest that the RNAs are relatively stable, perhaps in part because their great length and/or stem-and-loop structures at their 3' ends help to protect them against exonucleolytic degradation.
6,415 citations
"Protein production by auto-inductio..." refers background or methods in this paper
...T7 RNA polymerase is so active that induction can direct most transcription and translation to the target protein [1], which might interfere with full induction of the ability to metabolize lactose for energy....
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...DE3 lysogens contain a derivative of phage lambda that supplies T7 RNA polymerase by transcription from the lacUV5 promoter in the chromosome [1]....
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...Media previously described [1] for growth of E....
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...Such overgrowth can occur when expression vectors confer resistance to ampicillin, because secreted b-lactamase can degrade all of the ampicillin in the medium [1,2]....
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...Perhaps more important, induced T7 RNA polymerase can be so active that most transcription and protein synthesis in the cell is directed toward target protein [1]....
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TL;DR: The tight regulation of the PBAD promoter is exploited to study the phenotypes of null mutations of essential genes and the use of pBAD vectors as an expression system is explored.
Abstract: We have constructed a series of plasmid vectors (pBAD vectors) containing the PBAD promoter of the araBAD (arabinose) operon and the gene encoding the positive and negative regulator of this promoter, araC. Using the phoA gene and phoA fusions to monitor expression in these vectors, we show that the ratio of induction/repression can be 1,200-fold, compared with 50-fold for PTAC-based vectors. phoA expression can be modulated over a wide range of inducer (arabinose) concentrations and reduced to extremely low levels by the presence of glucose, which represses expression. Also, the kinetics of induction and repression are very rapid and significantly affected by the ara allele in the host strain. Thus, the use of this system which can be efficiently and rapidly turned on and off allows the study of important aspects of bacterial physiology in a very simple manner and without changes of temperature. We have exploited the tight regulation of the PBAD promoter to study the phenotypes of null mutations of essential genes and explored the use of pBAD vectors as an expression system.
4,997 citations
"Protein production by auto-inductio..." refers background in this paper
...Auto-induction with arabinose—Expression systems in which transcription is controlled by the pBAD promoter of the arabinose operon have relatively low basal expression, which can make them useful for maintaining and expressing toxic genes [38, 39]....
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Book•
01 Jun 1987
4,940 citations
"Protein production by auto-inductio..." refers background in this paper
...Several amino acids substantially increased the lag or decreased the growth rate in minimal LG medium, most notably serine, alanine, leucine, and valine, presumably by repressing overlapping metabolic pathways [27,28]....
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