An extensive plasmid library for preparing tau variants and studying their functional biochemistry

Abstract We describe a new approach for identifying the gene corresponding to a mutation in Saccharomyces cerevisiae. A library of mTn-lacZ/LEU2 insertions is tested for failure to complement the mutation, and the noncomplementing insertion is used to obtain sequence. This approach offers an alternative to cloning by complementation with a plasmid library.

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Transcriptional Regulators of theSchizosaccharomyces pombe fbp1Gene Include Two Redundant Tup1p-like Corepressors and the CCAAT Binding Factor Activation Complex

Genetics ◽

10.1093/genetics/157.3.1205 ◽

2001 ◽

Vol 157 (3) ◽

pp. 1205-1215 ◽

Cited By ~ 4

Author(s):

Rozmin T K Janoo ◽

Lori A Neely ◽

Burkhard R Braun ◽

Simon K Whitehall ◽

Charles S Hoffman

Keyword(s):

Protein Kinase ◽

Fold Increase ◽

Transcriptional Regulators ◽

Dominant Negative ◽

Mitogen Activated Protein Kinase ◽

Lacz Expression ◽

Protein Subunit ◽

Plasmid Library ◽

Binding Factor ◽

Mitogen Activated Protein

AbstractThe Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK). To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity. Two such clones express amino-terminally truncated forms of the S. pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor. These clones appear to act as dominant negative alleles. Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription. In strains lacking tup11 and tup12, the atf1-pcr1 transcriptional activator continues to play a central role in fbp1-lacZ expression; however, spc1 MAPK phosphorylation of atf1 is no longer essential for its activation. We discuss possible models for the role of tup11- and tup12-mediated repression with respect to signaling from the MAPK and PKA pathways. A third clone identified in our screen expresses the php5 protein subunit of the CCAAT-binding factor (CBF). Deletion of php5 reduces fbp1 expression under both repressed and derepressed conditions. The CBF appears to act in parallel to atf1-pcr1, although it is unclear whether or not CBF activity is regulated by PKA.

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Isolation and transcriptional characterization of three genes which function at start, the controlling event of the Saccharomyces cerevisiae cell division cycle: CDC36, CDC37, and CDC39

Molecular and Cellular Biology ◽

10.1128/mcb.3.5.881-891.1983 ◽

1983 ◽

Vol 3 (5) ◽

pp. 881-891

Author(s):

H J Breter ◽

J Ferguson ◽

T A Peterson ◽

S I Reed

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Division ◽

Dna Sequences ◽

Shuttle Vector ◽

Control Process ◽

Haploid Cell ◽

Loop Analysis ◽

Mrna Species ◽

Coding Regions ◽

Plasmid Library

The genes CDC36, CDC37, and CDC39, thought to function in the cell division control process in Saccharomyces cerevisiae, were isolated from a recombinant plasmid library prepared by partial digestion of S. cerevisiae genomic DNA with Sau3A and insertion into the S. cerevisiae-Escherichia coli shuttle vector YRp7. In each case, S. cerevisiae DNA sequences were identified which could complement mutant alleles of the gene in question and which could direct integration of a plasmid at the chromosomal location known to correspond to that gene. Complementing DNA segments were subcloned to remove extraneous coding regions. The coding regions corresponding to CDC36, CDC37, and CDC39 were then identified and localized by R-loop analysis. The estimated sizes of the three coding regions were 615, 1,400, and 2,700 base pairs, respectively. Transcriptional orientation of the coding regions was established by using M13 vectors to prepare strand-specific probes followed by hybridization to blots of electrophoresed S. cerevisiae mRNA. The intracellular steady-state abundance of the mRNA species corresponding to the genes was estimated by comparing hybridization signals on RNA blots to that of a previously determined standard, the cell cycle start gene CDC28. The quantities calculated for the three mRNA species were low, ranging from 1.5 +/- 1 copies per haploid cell for the CDC36 mRNA to 3.1 +/- 1.5 and 4.6 +/- 2 copies per haploid cell for the CDC37 and CDC39 mRNAs, respectively. The CDC28 mRNA had been previously estimated at 7.0 +/- 2 copies per cell.

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Metabolic engineering of Escherichia coli W for isobutanol production on chemically defined medium and cheese whey as alternative raw material

Journal of Industrial Microbiology & Biotechnology ◽

10.1007/s10295-020-02319-y ◽

2020 ◽

Vol 47 (12) ◽

pp. 1117-1132

Author(s):

Katharina Novak ◽

Juliane Baar ◽

Philipp Freitag ◽

Stefan Pflügl

Keyword(s):

Escherichia Coli ◽

Cheese Whey ◽

Strain Improvement ◽

Defined Medium ◽

Raw Material ◽

Efficient Production ◽

Substrate Uptake ◽

Chemically Defined Medium ◽

E Coli ◽

Plasmid Library

AbstractThe aim of this study was to establish isobutanol production on chemically defined medium in Escherichia coli. By individually expressing each gene of the pathway, we constructed a plasmid library for isobutanol production. Strain screening on chemically defined medium showed successful production in the robust E. coli W strain, and expression vector IB 4 was selected as the most promising construct due to its high isobutanol yields and efficient substrate uptake. The investigation of different aeration strategies in combination with strain improvement and the implementation of a pulsed fed-batch were key for the development of an efficient production process. E. coli W ΔldhA ΔadhE Δpta ΔfrdA enabled aerobic isobutanol production at 38% of the theoretical maximum. Use of cheese whey as raw material resulted in longer process stability, which allowed production of 20 g l−1 isobutanol. Demonstrating isobutanol production on both chemically defined medium and a residual waste stream, this study provides valuable information for further development of industrially relevant isobutanol production processes.

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Rapid optimisation of cellulolytic enzymes ratios in Saccharomyces cerevisiae using in vitro SCRaMbLE

Biotechnology for Biofuels ◽

10.1186/s13068-020-01823-8 ◽

2020 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Elizabeth L. I. Wightman ◽

Heinrich Kroukamp ◽

Isak S. Pretorius ◽

Ian T. Paulsen ◽

Helena K. M. Nevalainen

Keyword(s):

Saccharomyces Cerevisiae ◽

Enzyme Activities ◽

Consolidated Bioprocessing ◽

Synergistic Action ◽

Cellulosic Biomass ◽

Gene Copy ◽

Cellulolytic Enzymes ◽

Plasmid Library ◽

Substrate Hydrolysis

Abstract Background For the economic production of biofuels and other valuable products from lignocellulosic waste material, a consolidated bioprocessing (CBP) organism is required. With efficient fermentation capability and attractive industrial qualities, Saccharomyces cerevisiae is a preferred candidate and has been engineered to produce enzymes that hydrolyze cellulosic biomass. Efficient cellulose hydrolysis requires the synergistic action of several enzymes, with the optimum combined activity ratio dependent on the composition of the substrate. Results In vitro SCRaMbLE generated a library of plasmids containing different ratios of a β-glucosidase gene (CEL3A) from Saccharomycopsis fibuligera and an endoglucanase gene (CEL5A) from Trichoderma reesei. S. cerevisiae, transformed with the plasmid library, displayed a range of individual enzyme activities and synergistic capabilities. Furthermore, we show for the first time that 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-d-cellopentaoside (BPNPG5) is a suitable substrate to determine synergistic Cel3A and Cel5A action and an accurate predictive model for this synergistic action was devised. Strains with highest BPNPG5 activity had an average CEL3A and CEL5A gene cassette copy number of 1.3 ± 0.6 and 0.8 ± 0.2, respectively (ratio of 1.6:1). Conclusions Here, we describe a synthetic biology approach to rapidly optimise gene copy numbers to achieve efficient synergistic substrate hydrolysis. This study demonstrates how in vitro SCRaMbLE can be applied to rapidly combine gene constructs in various ratios to allow screening of synergistic enzyme activities for efficient substrate hydrolysis.

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Roles of Cyclic AMP Receptor Protein and the Carboxyl-Terminal Domain of the α Subunit in Transcription Activation of theEscherichia coli rhaBAD Operon

Journal of Bacteriology ◽

10.1128/jb.182.12.3529-3535.2000 ◽

2000 ◽

Vol 182 (12) ◽

pp. 3529-3535 ◽

Cited By ~ 22

Author(s):

Carolyn C. Holcroft ◽

Susan M. Egan

Keyword(s):

Dna Binding ◽

Cyclic Amp ◽

Full Length ◽

Receptor Protein ◽

Α Subunit ◽

Cyclic Amp Receptor Protein ◽

Terminal Domain ◽

Carboxyl Terminal Domain ◽

Plasmid Library ◽

Carboxyl Terminal

ABSTRACT The Escherichia coli rhaBAD operon encodes the enzymes for catabolism of the sugar l-rhamnose. FullrhaBAD activation requires the AraC family activator RhaS (bound to a site that overlaps the −35 region of the promoter) and the cyclic AMP receptor protein (CRP; bound immediately upstream of RhaS at −92.5). We tested alanine substitutions in activating regions (AR) 1 and 2 of CRP for their effect onrhaBAD activation. Some, but not all, of the substitutions in both AR1 and AR2 resulted in approximately twofold defects in expression from rhaBAD promoter fusions. We also expressed a derivative of the α subunit of RNA polymerase deleted for the entire C-terminal domain (α-Δ235) and assayed expression from rhaBAD promoter fusions. The greatest defect (54-fold) occurred at a truncated promoter where RhaS was the only activator, while the defect at the full-length promoter (RhaS plus CRP) was smaller (13-fold). Analysis of a plasmid library expressing alanine substitutions at every residue in the carboxyl-terminal domain of the α subunit (α-CTD) identified 15 residues (mostly in the DNA-binding determinant) that were important at both the full-length and truncated promoters. Only one substitution was defective at the full-length but not the truncated promoter, and this residue was located in the DNA-binding determinant. Six substitutions were defective only at the promoter activated by RhaS alone, and these may define a protein-contacting determinant on α-CTD. Overall, our results suggest that CRP interaction with α-CTD may not be required for rhaBAD activation; however, α-CTD does contribute to full activation, probably through interactions with DNA and possibly RhaS.

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A Rapid, Simple, and Effective Method of Constructing a Randomly Mutagenized Plasmid Library Free from Ligation

Bioscience Biotechnology and Biochemistry ◽

10.1271/bbb.70814 ◽

2008 ◽

Vol 72 (4) ◽

pp. 1134-1137 ◽

Cited By ~ 6

Author(s):

Takashi KOYANAGI ◽

Erina YOSHIDA ◽

Hiromichi MINAMI ◽

Takane KATAYAMA ◽

Hidehiko KUMAGAI

Keyword(s):

Plasmid Library

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A universal plasmid library encoding all permutations of small interfering RNA

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0401549101 ◽

2005 ◽

Vol 102 (7) ◽

pp. 2356-2361 ◽

Cited By ~ 32

Author(s):

M. Chen ◽

L. Zhang ◽

H.-Y. Zhang ◽

X. Xiong ◽

B. Wang ◽

...

Keyword(s):

Small Interfering Rna ◽

Plasmid Library ◽

Interfering Rna

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Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

Journal of Clinical Microbiology ◽

10.1128/jcm.02104-16 ◽

2017 ◽

Vol 55 (6) ◽

pp. 1755-1766 ◽

Cited By ~ 9

Author(s):

Divya Anthwal ◽

Rakesh Kumar Gupta ◽

Manpreet Bhalla ◽

Shinjini Bhatnagar ◽

Jaya Sivaswami Tyagi ◽

...

Keyword(s):

High Resolution ◽

Direct Detection ◽

Melting Curve ◽

Curve Analysis ◽

High Resolution Melt ◽

Melt Curve Analysis ◽

Content Type ◽

Plasmid Library ◽

Mdr Tb ◽

Smear Negative

ABSTRACT Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB , katG , and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH- katG , and INH- inhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% (κ value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR]400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.

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The Novel Transcription Factor SgrR Coordinates the Response to Glucose-Phosphate Stress

Journal of Bacteriology ◽

10.1128/jb.01689-06 ◽

2007 ◽

Vol 189 (6) ◽

pp. 2238-2248 ◽

Cited By ~ 73

Author(s):

Carin K. Vanderpool ◽

Susan Gottesman

Keyword(s):

Binding Site ◽

Binding Domain ◽

Alternative Mechanism ◽

Gene Encoding ◽

Bacterial Transcription ◽

Glucose Phosphate ◽

Plasmid Library ◽

Promoter Dna

ABSTRACT SgrR is the first characterized member of a family of bacterial transcription factors containing an N-terminal DNA binding domain and a C-terminal solute binding domain. Previously, we reported genetic evidence that SgrR activates the divergently transcribed gene sgrS, which encodes a small RNA required for recovery from glucose-phosphate stress. In this study, we examined the regulation of sgrR expression and found that SgrR negatively autoregulates its own transcription in the presence and absence of stress. An SgrR binding site in the sgrR-sgrS intergenic region is required in vivo for both SgrR-dependent activation of sgrS and autorepression of sgrR. Purified SgrR binds specifically to sgrS promoter DNA in vitro; a mutation in the site required for in vivo activation and autorepression abrogates in vitro SgrR binding. A plasmid library screen identified clones that alter expression of a P sgrS -lacZ fusion; some act by titrating endogenous SgrR. The yfdZ gene, encoding a putative aminotransferase, was identified in this screen; the yfdZ promoter contains an SgrR binding site, and transcriptional fusions indicate that yfdZ is activated by SgrR. Clones containing mlc, which encodes a glucose-specific repressor protein, also downregulate P sgrS -lacZ. The mlc clones do not appear to titrate the SgrR protein, indicating that Mlc affects sgrS expression by an alternative mechanism.

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