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A standardized workflow for surveying recombinases expands bacterial genome-editing capabilities.

ABSTRACT: Bacterial recombineering typically relies on genomic incorporation of synthetic oligonucleotides as mediated by Escherichia coli ? phage recombinase ? - an occurrence largely limited to enterobacterial strains. While a handful of similar recombinases have been documented, recombineering efficiencies usually fall short of expectations for practical use. In this work, we aimed to find an efficient Rec? homologue demonstrating activity in model soil bacterium Pseudomonas putida EM42. To this end, a genus-wide protein survey was conducted to identify putative recombinase candidates for study. Selected novel proteins were assayed in a standardized test to reveal their ability to introduce the K43T substitution into the rpsL gene of P. putida. An ERF superfamily protein, here termed Rec2, exhibited activity eightfold greater than that of the previous leading recombinase. To bolster these results, we demonstrated Rec2 ability to enter a range of mutations into the pyrF gene of P. putida at similar frequencies. Our results not only confirm the utility of Rec2 as a Rec? functional analogue within the P. putida model system, but also set a complete workflow for deploying recombineering in other bacterial strains/species. Implications range from genome editing of P. putida for metabolic engineering to extended applications within other Pseudomonads - and beyond.

SUBMITTER: Ricaurte DE

PROVIDER: S-EPMC5743808 | biostudies-other | 2018 Jan

REPOSITORIES: biostudies-other

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A standardized workflow for surveying recombinases expands bacterial genome-editing capabilities.

Ricaurte Deirdre E DE Martínez-García Esteban E Nyerges Ákos Á Pál Csaba C de Lorenzo Víctor V Aparicio Tomás T

Microbial biotechnology 20171102 1

Bacterial recombineering typically relies on genomic incorporation of synthetic oligonucleotides as mediated by Escherichia coli λ phage recombinase β - an occurrence largely limited to enterobacterial strains. While a handful of similar recombinases have been documented, recombineering efficiencies usually fall short of expectations for practical use. In this work, we aimed to find an efficient Recβ homologue demonstrating activity in model soil bacterium Pseudomonas putida EM42. To this end, a ...[more]

PMID: 29094478

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Project description:PurposeMotion management of tumors within the lung and abdomen is challenging because it requires balancing tissue sparing with accuracy of hitting the target, while considering treatment delivery efficiency. Physicists can play an important role in analyzing four-dimensional computed tomography (4DCT) data to recommend the optimal respiratory gating parameters for a patient. The goal of this work was to develop a standardized procedure for making recommendations regarding gating parameters and planning margins for lung and gastrointestinal stereotactic body radiotherapy (SBRT) treatments. In doing so, we hoped to simplify decision-making and analysis, and provide a tool for troubleshooting complex cases.MethodsFactors that impact gating decisions and planning target volume (PTV) margins were identified. The gating options included gating on exhale with approximately a 50% duty cycle (Gate3070), exhale gating with a reduced duty cycle (Gate4060), and treating for most of respiration, excluding only extreme inhales and exhales (Gate100). A standard operating procedure was developed, as well as a physics consult document to communicate motion management recommendations to other members of the treatment team. This procedure was implemented clinically for 1 year and results are reported below.ResultsIdentified factors that impact motion management included the magnitude of motion observed on 4DCT, the regularity of breathing and quality of 4DCT data, and ability to observe the target on fluoroscopy. These were collated into two decision tables-one specific to lung tumors and another for gastrointestinal tumors-such that a physicist could answer a series of questions to determine the optimal gating and PTV margin. The procedure was used clinically for 252 sites from 213 patients treated with respiratory-gated SBRT and standardized practice across our 12-member physics team.ConclusionImplementation of a standardized procedure for respiratory gating had a positive impact in our clinic, improving efficiency and ease of 4DCT analysis and standardizing gating decision-making amongst physicists.

Dataset Information

A standardized workflow for surveying recombinases expands bacterial genome-editing capabilities.

Publications

A standardized workflow for surveying recombinases expands bacterial genome-editing capabilities.

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