scholarly journals Differentially optimized cell-free buffer enables robust expression from unprotected linear DNA in exonuclease-deficient extracts

2021 ◽  
Author(s):  
Angelo Cardoso Batista ◽  
Antoine Levrier ◽  
Paul Soudier ◽  
Peter Voyvodic ◽  
Tatjana Achmedov ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Additional Time ◽  
Experimental Conditions ◽  
Dna Templates ◽  
Linear Dna ◽  
Synthetic Gene Circuits ◽  
Cell Extracts ◽  
Wide Range ◽  
Additional Protection ◽  
Protection Strategies

ABSTRACTThe use of linear DNA templates in cell-free systems promises to accelerate the prototyping and engineering of synthetic gene circuits. A key challenge is that linear templates are rapidly degraded by exonucleases present in cell extracts. Current approaches tackle the problem by adding exonuclease inhibitors and DNA-binding proteins to protect the linear DNA, requiring additional time- and resource-intensive steps. Here, we delete the recBCD exonuclease gene cluster from the Escherichia coli BL21 genome. We show that the resulting cell-free systems, with buffers optimized specifically for linear DNA, enable near-plasmid levels of expression from σ70 promoters in linear DNA templates without employing additional protection strategies. When using linear or plasmid DNA templates at the buffer calibration step, we found that the optimal potassium glutamate concentrations obtained when using linear DNA were consistently lower than those obtained when using plasmid DNA for the same extract. We demonstrate the robustness of the exonuclease deficient extracts across seven different batches and a wide range of experimental conditions. Finally, we illustrate the use of the ΔrecBCD extracts for two applications: toehold switch characterization and enzyme screening. Our work provides a simple, efficient, and costeffective solution for using linear DNA templates in cell-free systems and highlights the importance of specifically tailoring buffer composition for the final experimental setup. Our data also suggest that similar exonuclease deletion strategies can be applied to other species suitable for cell-free synthetic biology.

scholarly journals In vitro cell cycle arrest induced by using artificial DNA templates

1992 ◽  
Vol 12 (7) ◽  
pp. 3216-3223
Author(s):  
S Kornbluth ◽  
C Smythe ◽  
J W Newport
Keyword(s):  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Plasmid Dna ◽  
Signal Transduction Pathway ◽  
Dna Templates ◽  
Single Stranded Dna ◽  
Cell Extracts ◽  
Artificial Dna ◽  
Dna Template

In cell extracts of Xenopus eggs which oscillate between S and M phases of the cell cycle, the onset of mitosis is blocked by the presence of incompletely replicated DNA. In this report, we show that several artificial DNA templates (M13 single-stranded DNA and double-stranded plasmid DNA) can trigger this feedback pathway, which inhibits mitosis. Single-stranded M13 DNA is much more effective than double-stranded plasmid DNA at inhibiting the onset of mitosis. Furthermore, we have shown that low levels of M13 single-stranded DNA and high levels of double-stranded plasmid DNA can elevate the tyrosine kinase activity responsible for phosphorylating p34cdc2, thereby inactivating maturation-promoting factor and inhibiting entry into mitosis. This constitutes a simplified system with which to study the signal transduction pathway from the DNA template to the tyrosine kinase responsible for inhibiting p34cdc2 activity.

scholarly journals In vitro cell cycle arrest induced by using artificial DNA templates.

1992 ◽  
Vol 12 (7) ◽  
pp. 3216-3223 ◽  
Author(s):  
S Kornbluth ◽  
C Smythe ◽  
J W Newport
Keyword(s):  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Plasmid Dna ◽  
Signal Transduction Pathway ◽  
Dna Templates ◽  
Single Stranded Dna ◽  
Cell Extracts ◽  
Artificial Dna ◽  
Dna Template

In cell extracts of Xenopus eggs which oscillate between S and M phases of the cell cycle, the onset of mitosis is blocked by the presence of incompletely replicated DNA. In this report, we show that several artificial DNA templates (M13 single-stranded DNA and double-stranded plasmid DNA) can trigger this feedback pathway, which inhibits mitosis. Single-stranded M13 DNA is much more effective than double-stranded plasmid DNA at inhibiting the onset of mitosis. Furthermore, we have shown that low levels of M13 single-stranded DNA and high levels of double-stranded plasmid DNA can elevate the tyrosine kinase activity responsible for phosphorylating p34cdc2, thereby inactivating maturation-promoting factor and inhibiting entry into mitosis. This constitutes a simplified system with which to study the signal transduction pathway from the DNA template to the tyrosine kinase responsible for inhibiting p34cdc2 activity.

scholarly journals In silico Design of Linear DNA for Robust Cell-Free Gene Expression

2021 ◽  
Vol 9 ◽  
Author(s):  
Xinjie Chen ◽  
Yuan Lu
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
High Throughput Screening ◽  
Expression System ◽  
Dna Templates ◽  
Linear Dna ◽  
Low Protein ◽  
Cell Extracts ◽  
Free Gene ◽  
Expression Templates

Cell-free gene expression systems with linear DNA expression templates (LDETs) have been widely applied in artificial cells, biochips, and high-throughput screening. However, due to the degradation caused by native nucleases in cell extracts, the transcription with linear DNA templates is weak, thereby resulting in low protein expression level, which greatly limits the development of cell-free systems using linear DNA templates. In this study, the protective sequences for stabilizing linear DNA and the transcribed mRNAs were rationally designed according to nucleases’ action mechanism, whose effectiveness was evaluated through computer simulation and cell-free gene expression. The cell-free experiment results indicated that, with the combined protection of designed sequence and GamS protein, the protein expression of LDET-based cell-free systems could reach the same level as plasmid-based cell-free systems. This study would potentially promote the development of the LDET-based cell-free gene expression system for broader applications.

In vivo Production of Soluble Inorganic Pyrophosphatases in Two Strains of Vibrio cholerae

1970 ◽  
Vol 24 (1) ◽  
pp. 38-41
Author(s):  
Taslima Taher Lina ◽  
Mohammad Ilias
Keyword(s):  
Vibrio Cholerae ◽  
Specific Activity ◽  
Experimental Conditions ◽  
Environmental Strain ◽  
Cell Extracts ◽  
Atcc Strain ◽  
The Family ◽  
Effects Of Ph ◽  
Vibrio Cholerae O1

The in vivo production of soluble inorganic pyrophosphatases (PPases) was investigated in two strains, namely, Vibrio cholerae EM 004 (environmental strain) and Vibrio cholerae O1 757 (ATCC strain). V. cholerae is known to contain both family I and family II PPase coding sequences. The production of family I and family II PPases were determined by measuring the enzyme activity in cell extracts. The effects of pH, temperature, salinity of the growth medium on the production of soluble PPases were studied. In case of family I PPase, V. cholerae EM 004 gave the highest specific activity at pH 9.0, with 2% NaCl + 0.011% NaF and at 37°C. The strain V. cholerae O1 757 gave the highest specific activity at pH 9.0, with media containing 0% NaCl and at 37°C. On the other hand, under all the conditions family II PPase did not give any significant specific activity, suggesting that the family II PPase was not produced in vivo in either strains of V. cholerae under different experimental conditions. Keywords: Vibrio cholerae, Pyrophosphatases (PPases), Specific activityDOI: http://dx.doi.org/10.3329/bjm.v24i1.1235 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 38-41

Strongly Coupled Redox-Linked Conformational Switching at the Active Site of the Non-Heme Iron-Dependent Dioxygenase, TauD

2019 ◽  
Author(s):  
Christopher John ◽  
Greg M. Swain ◽  
Robert P. Hausinger ◽  
Denis A. Proshlyakov
Keyword(s):  
Active Site ◽  
Structural Model ◽  
Heme Iron ◽  
Chemical Transformations ◽  
Experimental Conditions ◽  
Strongly Coupled ◽  
Non Heme Iron ◽  
Reversible Redox ◽  
Wide Range ◽  
Complex Structural

2-Oxoglutarate (2OG)-dependent dioxygenases catalyze C-H activation while performing a wide range of chemical transformations. In contrast to their heme analogues, non-heme iron centers afford greater structural flexibility with important implications for their diverse catalytic mechanisms. We characterize an <i>in situ</i> structural model of the putative transient ferric intermediate of 2OG:taurine dioxygenase (TauD) by using a combination of spectroelectrochemical and semi-empirical computational methods, demonstrating that the Fe (III/II) transition involves a substantial, fully reversible, redox-linked conformational change at the active site. This rearrangement alters the apparent redox potential of the active site between -127 mV for reduction of the ferric state and 171 mV for oxidation of the ferrous state of the 2OG-Fe-TauD complex. Structural perturbations exhibit limited sensitivity to mediator concentrations and potential pulse duration. Similar changes were observed in the Fe-TauD and taurine-2OG-Fe-TauD complexes, thus attributing the reorganization to the protein moiety rather than the cosubstrates. Redox difference infrared spectra indicate a reorganization of the protein backbone in addition to the involvement of carboxylate and histidine ligands. Quantitative modeling of the transient redox response using two alternative reaction schemes across a variety of experimental conditions strongly supports the proposal for intrinsic protein reorganization as the origin of the experimental observations.

scholarly journals Transfer of Plasmid DNA to Clinical Coagulase-Negative Staphylococcal Pathogens by Using a Unique Bacteriophage

2015 ◽  
Vol 81 (7) ◽  
pp. 2481-2488 ◽  
Author(s):  
Volker Winstel ◽  
Petra Kühner ◽  
Bernhard Krismer ◽  
Andreas Peschel ◽  
Holger Rohde
Keyword(s):  
Plasmid Dna ◽  
Genetic Manipulation ◽  
Current Knowledge ◽  
Virulence Determinants ◽  
Coagulase Negative Staphylococci ◽  
Reliable Technique ◽  
Content Type ◽  
Research Activities ◽  
Wide Range ◽  
Genetic Barriers

ABSTRACTGenetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a uniqueStaphylococcus aureusstrain via a specificS. aureusbacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinicalStaphylococcus epidermidisisolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

scholarly journals Low-Energy Electron Damage to Condensed-Phase DNA and Its Constituents

2021 ◽  
Vol 22 (15) ◽  
pp. 7879
Author(s):  
Yingxia Gao ◽  
Yi Zheng ◽  
Léon Sanche
Keyword(s):  
Condensed Phase ◽  
Genetic Material ◽  
High Energy ◽  
Dna Lesions ◽  
Low Energy ◽  
Experimental Investigations ◽  
Experimental Conditions ◽  
Strand Breaks ◽  
Sequence Of Events ◽  
Wide Range

The complex physical and chemical reactions between the large number of low-energy (0–30 eV) electrons (LEEs) released by high energy radiation interacting with genetic material can lead to the formation of various DNA lesions such as crosslinks, single strand breaks, base modifications, and cleavage, as well as double strand breaks and other cluster damages. When crosslinks and cluster damages cannot be repaired by the cell, they can cause genetic loss of information, mutations, apoptosis, and promote genomic instability. Through the efforts of many research groups in the past two decades, the study of the interaction between LEEs and DNA under different experimental conditions has unveiled some of the main mechanisms responsible for these damages. In the present review, we focus on experimental investigations in the condensed phase that range from fundamental DNA constituents to oligonucleotides, synthetic duplex DNA, and bacterial (i.e., plasmid) DNA. These targets were irradiated either with LEEs from a monoenergetic-electron or photoelectron source, as sub-monolayer, monolayer, or multilayer films and within clusters or water solutions. Each type of experiment is briefly described, and the observed DNA damages are reported, along with the proposed mechanisms. Defining the role of LEEs within the sequence of events leading to radiobiological lesions contributes to our understanding of the action of radiation on living organisms, over a wide range of initial radiation energies. Applications of the interaction of LEEs with DNA to radiotherapy are briefly summarized.

scholarly journals Review of the Upright Balance Assessment Based on the Force Plate

2021 ◽  
Vol 18 (5) ◽  
pp. 2696
Author(s):  
Baoliang Chen ◽  
Peng Liu ◽  
Feiyun Xiao ◽  
Zhengshi Liu ◽  
Yong Wang
Keyword(s):  
Postural Balance ◽  
Force Plate ◽  
Assessment Method ◽  
Balance Assessment ◽  
Experimental Conditions ◽  
Methodological Research ◽  
Assessment Tests ◽  
Wide Range ◽  
Foot Posture ◽  
Plate System

Quantitative assessment is crucial for the evaluation of human postural balance. The force plate system is the key quantitative balance assessment method. The purpose of this study is to review the important concepts in balance assessment and analyze the experimental conditions, parameter variables, and application scope based on force plate technology. As there is a wide range of balance assessment tests and a variety of commercial force plate systems to choose from, there is room for further improvement of the test details and evaluation variables of the balance assessment. The recommendations presented in this article are the foundation and key part of the postural balance assessment; these recommendations focus on the type of force plate, the subject’s foot posture, and the choice of assessment variables, which further enriches the content of posturography. In order to promote a more reasonable balance assessment method based on force plates, further methodological research and a stronger consensus are still needed.

scholarly journals Excision of Oxidatively Generated Guanine Lesions by Competitive DNA Repair Pathways

2021 ◽  
Vol 22 (5) ◽  
pp. 2698
Author(s):  
Vladimir Shafirovich ◽  
Nicholas E. Geacintov
Keyword(s):  
Excision Repair ◽  
Dna Lesions ◽  
Dna Templates ◽  
Circular Dna ◽  
Damage Sensing ◽  
Cell Extracts ◽  
Dna Base ◽  
Nucleotide Excision ◽  
Repair Pathways ◽  
Cellular Dna

The base and nucleotide excision repair pathways (BER and NER, respectively) are two major mechanisms that remove DNA lesions formed by the reactions of genotoxic intermediates with cellular DNA. It is generally believed that small non-bulky oxidatively generated DNA base modifications are removed by BER pathways, whereas DNA helix-distorting bulky lesions derived from the attack of chemical carcinogens or UV irradiation are repaired by the NER machinery. However, existing and growing experimental evidence indicates that oxidatively generated DNA lesions can be repaired by competitive BER and NER pathways in human cell extracts and intact human cells. Here, we focus on the interplay and competition of BER and NER pathways in excising oxidatively generated guanine lesions site-specifically positioned in plasmid DNA templates constructed by a gapped-vector technology. These experiments demonstrate a significant enhancement of the NER yields in covalently closed circular DNA plasmids (relative to the same, but linearized form of the same plasmid) harboring certain oxidatively generated guanine lesions. The interplay between the BER and NER pathways that remove oxidatively generated guanine lesions are reviewed and discussed in terms of competitive binding of the BER proteins and the DNA damage-sensing NER factor XPC-RAD23B to these lesions.

scholarly journals Towards Reduced CNNs for De-Noising Phase Images Corrupted with Speckle Noise

Photonics ◽  
2021 ◽  
Vol 8 (7) ◽  
pp. 255
Author(s):  
Marie Tahon ◽  
Silvio Montresor ◽  
Pascal Picart
Keyword(s):  
Phase Error ◽  
Speckle Noise ◽  
Experimental Conditions ◽  
Benchmark Data ◽  
Wide Range ◽  
Phase Images ◽  
Different Depths ◽  
Phase Data ◽  
Do So

Digital holography is a very efficient technique for 3D imaging and the characterization of changes at the surfaces of objects. However, during the process of holographic interferometry, the reconstructed phase images suffer from speckle noise. In this paper, de-noising is addressed with phase images corrupted with speckle noise. To do so, DnCNN residual networks with different depths were built and trained with various holographic noisy phase data. The possibility of using a network pre-trained on natural images with Gaussian noise is also investigated. All models are evaluated in terms of phase error with HOLODEEP benchmark data and with three unseen images corresponding to different experimental conditions. The best results are obtained using a network with only four convolutional blocks and trained with a wide range of noisy phase patterns.

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