scholarly journals Model-driven generation of artificial yeast promoters

2019 ◽  
Author(s):  
Benjamin J. Kotopka ◽  
Christina D. Smolke
Keyword(s):  
Model Organism ◽  
Inducible Promoter ◽  
Design Strategies ◽  
Multiple Sequence ◽  
Promoter Sequences ◽  
Model Driven ◽  
Large Sets ◽  
Promoter Library ◽  
Small Set ◽  
Rapid Generation

AbstractPromoters play a central role in controlling gene regulation; however, a small set of promoters is used for most genetic construct design in the yeast Saccharomyces cerevisiae. Generating and utilizing models that accurately predict protein expression from promoter sequences would enable rapid generation of novel useful promoters and facilitate synthetic biology efforts in this model organism. We measured the gene expression activity of over 675,000 unique sequences in a constitutive promoter library, and over 327,000 sequences in an inducible promoter library. Training an ensemble of convolutional neural networks jointly on the two datasets enabled very high (R2 > 0.79) predictive accuracies on multiple sequence-activity prediction tasks. We developed model-guided design strategies which yielded large, sequence-diverse sets of novel promoters exhibiting activities similar to current best-in-class sequences. In addition to providing large sets of new promoters, our results show the value of model-guided design as an approach for generating useful DNA parts.

scholarly journals Model-driven promoter strength prediction based on a fine-tuned synthetic promoter library in Escherichia coli

2020 ◽  
Author(s):  
Mei Zhao ◽  
Shenghu Zhou ◽  
Longtao Wu ◽  
Yu Deng
Keyword(s):  
Metabolic Pathways ◽  
Regulatory Elements ◽  
Strength Prediction ◽  
Promoter Strength ◽  
Synthetic Promoter ◽  
Promoter Sequences ◽  
Model Driven ◽  
Promoter Library ◽  
Mutant Promoter ◽  
The Relationship

AbstractPromoters are one of the most critical regulatory elements controlling metabolic pathways. However, in recent years, researchers have simply perfected promoter strength, but ignored the relationship between the internal sequences and promoter strength. In this context, we constructed and characterized a mutant promoter library of Ptrc through dozens of mutation-construction-screening-characterization engineering cycles. After excluding invalid mutation sites, we established a synthetic promoter library, which consisted of 3665 different variants, displaying an intensity range of more than two orders of magnitude. The strongest variant was 1.52-fold stronger than a 1 mM isopropyl-β-D-thiogalactoside driven PT7 promoter. Our synthetic promoter library exhibited superior applicability when expressing different reporters, in both plasmids and the genome. Different machine learning models were built and optimized to explore relationships between the promoter sequences and transcriptional strength. Finally, our XgBoost model exhibited optimal performance, and we utilized this approach to precisely predict the strength of artificially designed promoter sequences. Our work provides a powerful platform that enables the predictable tuning of promoters to achieve the optimal transcriptional strength.

Synergistic Experimental and Computational Approach Identifies Novel Strategies for PHB Overproduction

2021 ◽  
Author(s):  
Adil Alsiyabi ◽  
Brandi Brown ◽  
Cheryl Immethun ◽  
Mark Wilkins ◽  
Rajib Saha
Keyword(s):  
Engineering Design ◽  
Rhodopseudomonas Palustris ◽  
Maximum Yield ◽  
Computational Approach ◽  
Coniferyl Alcohol ◽  
Breakdown Product ◽  
Design Strategies ◽  
Metabolic Factors ◽  
Model Driven ◽  
Phb Production

Abstract Polyhydroxybutyrate (PHB) is a sustainable bioplastic produced by bacteria that is a potential replacement for conventional plastics. This study delivers an integrated experimental and computational modeling approach to decipher metabolic factors controlling PHB production and offers engineering design strategies to boost production. In the metabolically robust Rhodopseudomonas palustris CGA009, PHB production significantly increased when grown on the carbon- and electron-rich lignin breakdown product p-coumarate (C9H8O3) compared to acetate when the same amount of carbon was supplied. However, the maximum yield did not improve further when grown on coniferyl alcohol (C10H12O3). In order to obtain a systems-level understanding of factors driving PHB yield, a model-driven investigation was performed. The model yielded several engineering design strategies including utilizing reduced, high molecular weight substrates that bypass the thiolase reaction. Overall, these findings uncover key parameters controlling PHB production and design strategies that can potentially be expanded to any bacterium for optimizing PHB production.

Adenovirus 5 E2 transcription unit: an E1A-inducible promoter with an essential element that functions independently of position or orientation

1984 ◽  
Vol 4 (5) ◽  
pp. 875-882
Author(s):  
M J Imperiale ◽  
J R Nevins
Keyword(s):  
Transcription Initiation ◽  
Essential Element ◽  
Inducible Promoter ◽  
Initiation Site ◽  
Transcription Unit ◽  
Upstream Sequence ◽  
Wild Type ◽  
Promoter Sequences ◽  
Adenovirus 5 ◽  
Wild Type Promoter

Utilizing deletion mutants of a plasmid containing the adenovirus E2 gene, an E1A-inducible transcription unit, we determined the promoter sequences required for full expression in transient transfection assays. Wild-type expression was obtained from plasmids containing only 79 nucleotides of upstream sequence relative to the transcription initiation site. Removal of an additional nine nucleotides lowered expression 10-fold, and deletion to -59 resulted in near total loss of transcription. Wild-type levels of expression were restored to a -28 deletion mutant by insertion of the sequence from -21 to -262 from the wild-type promoter at the -28 position, in either orientation, even though when inserted in the opposite orientation the relevant sequences were ca. 270 nucleotides upstream from their normal position. Finally, this sequence could be placed at a distance of 4,000 nucleotides from the E2 cap site and still retain near total function. Thus, the E2 promoter element can function independent of orientation and position, properties characteristic of enhancer elements.

scholarly journals A reversibly induced CRISPRi system targeting Photosystem II in the cyanobacterium Synechocystis sp. PCC 6803

2020 ◽  
Author(s):  
Deng Liu ◽  
Virginia M. Johnson ◽  
Himadri B. Pakrasi
Keyword(s):  
Photosystem Ii ◽  
Sole Carbon Source ◽  
Model Organism ◽  
Inducible Promoter ◽  
New Strategy ◽  
Photosynthetic Complexes ◽  
Promoter System ◽  
Synechocystis Sp ◽  
D2 Protein ◽  
Pcc 6803

ABSTRACTThe cyanobacterium Synechocystis sp. PCC 6803 is used as a model organism to study photosynthesis, as it can utilize glucose as the sole carbon source to support its growth under heterotrophic conditions. CRISPR interference (CRISPRi) has been widely applied to repress the transcription of genes in a targeted manner in cyanobacteria. However, a robust and reversible induced CRISPRi system has not been explored in Synechocystis 6803 to knock down and recover the expression of a targeted gene. In this study, we built a tightly controlled chimeric promoter, PrhaBAD-RSW, in which a theophylline responsive riboswitch was integrated into a rhamnose-inducible promoter system. We applied this promoter to drive the expression of ddCpf1 (DNase-dead Cpf1 nuclease) in a CRISPRi system and chose the PSII reaction center gene psbD (D2 protein) to target for repression. psbD was specifically knocked down by over 95% of its native expression, leading to severely inhibited Photosystem II activity and growth of Synechocystis 6803 under photoautotrophic conditions. Significantly, removal of the inducers rhamnose and theophylline reversed repression by CRISPRi. Expression of PsbD recovered following release of repression, coupled with increased Photosystem II content and activity. This reversibly induced CRISPRi system in Synechocystis 6803 represents a new strategy for study of the biogenesis of photosynthetic complexes in cyanobacteria.

scholarly journals A benchmark for evaluation of phylogeny reconstruction programs

2016 ◽  
Author(s):  
Sergei Spirin
Keyword(s):  
Maximum Likelihood ◽  
Sequence Alignment ◽  
Multiple Sequence Alignment ◽  
Protein Sequences ◽  
Relative Accuracy ◽  
Phylogeny Reconstruction ◽  
Multiple Sequence ◽  
Natural Protein ◽  
Large Sets ◽  
The Moment

There are a lot of algorithms and programs for reconstruction of phylogeny of a set of proteins basing on multiple sequence alignment. Many programs allow users to choose a number of parameters, for example, a model for maximum likelihood programs. Different programs and different parameters often produce different results. However at the moment all published benchmarks for evaluation of relative accuracy of programs or different choices of parameters are based on simulated sequences. The aim of the present work is to create a benchmark that allows a comparison of phylogenetic programs on large sets of alignments of natural protein sequences.

scholarly journals Rapid generation of pigment free, immobile zebrafish embryos and larvae in any genetic background using CRISPR-Cas9 dgRNPs

2021 ◽  
Author(s):  
Andrew E Davis ◽  
Daniel Castranova ◽  
Brant M. Weinstein
Keyword(s):  
High Resolution ◽  
Model Organism ◽  
Zebrafish Embryos ◽  
Pigment Formation ◽  
Developmental Defects ◽  
Chemical Treatments ◽  
Resolution Imaging ◽  
Rapid Generation ◽  
The Many ◽  
Mutant Lines

The ability to carry out high-resolution, high-magnification optical imaging of living animals is one of the most attractive features of the zebrafish as a model organism. However, formation of obscuring pigmentation as development proceeds and difficulties in maintaining sustained immobilization of healthy, living animals remain challenges that limit the application of live imaging. Chemical treatments can be used to suppress pigment formation and movement, but these treatments can lead to developmental defects. Genetic mutants can also be used to eliminate pigment formation and immobilize animals but maintaining these mutants in lines carrying other combinations of transgenes and mutants is difficult and laborious. Here, we show that CRISPR duplex guide ribonucleoproteins (dgRNPs) targeting the slc45a2 (albino) and chrna1 (nic1) genes can be used to efficiently suppress pigment formation in and immobilize F0 injected animals. CRISPR dgRNPs can be used to generate pigment-free, immobile zebrafish embryos and larvae in any transgenic and/or mutant-carrying background, greatly facilitating high-resolution imaging and analysis of the many transgenic and mutant lines available in the zebrafish.

scholarly journals Adenovirus 5 E2 transcription unit: an E1A-inducible promoter with an essential element that functions independently of position or orientation.

1984 ◽  
Vol 4 (5) ◽  
pp. 875-882 ◽  
Author(s):  
M J Imperiale ◽  
J R Nevins
Keyword(s):  
Transcription Initiation ◽  
Essential Element ◽  
Inducible Promoter ◽  
Initiation Site ◽  
Transcription Unit ◽  
Upstream Sequence ◽  
Wild Type ◽  
Promoter Sequences ◽  
Adenovirus 5 ◽  
Wild Type Promoter

Utilizing deletion mutants of a plasmid containing the adenovirus E2 gene, an E1A-inducible transcription unit, we determined the promoter sequences required for full expression in transient transfection assays. Wild-type expression was obtained from plasmids containing only 79 nucleotides of upstream sequence relative to the transcription initiation site. Removal of an additional nine nucleotides lowered expression 10-fold, and deletion to -59 resulted in near total loss of transcription. Wild-type levels of expression were restored to a -28 deletion mutant by insertion of the sequence from -21 to -262 from the wild-type promoter at the -28 position, in either orientation, even though when inserted in the opposite orientation the relevant sequences were ca. 270 nucleotides upstream from their normal position. Finally, this sequence could be placed at a distance of 4,000 nucleotides from the E2 cap site and still retain near total function. Thus, the E2 promoter element can function independent of orientation and position, properties characteristic of enhancer elements.

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