Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

CFE no Mn, with NADrec

ABSTRACT: CFE no Mn, with NADrec

SUBMITTER: Jacky Snoep

PROVIDER: 613 | FAIRDOMHub | 2020-01-06

REPOSITORIES: FAIRDOMHub

ACCESS DATA

Json Xml

Similar Datasets

A cell-free strategy for profiling intracellular antibiotic sensitivity and resistance – characterising the proteomes of K. pneumoniae ATCC 13882, ST258-T1b and NJST258-1 and E. coli K-12 MG1655.

Project description:Antimicrobial resistance (AMR) is a pandemic spread across multiple infectious disease microbes. To provide a new tool to study AMR, here we develop a Klebsiella pneumoniae cell-free gene expression (CFE) system. To characterise the system, we use proteomics to compare this to a Escherichia coli MG1655 CFE model, to identify relative differences and unique proteins. Then we use this native CFE system to profile antimicrobial activity in comparison to whole cell inhibition, to reveal host differences in IC50/MIC50 values. Finally, we use the CFE tool to study AMR variants, at a proof-of-concept level. As an exemplar, we show that RpoB H526L confers a 58-fold increase in CFE resistance to rifampicin – a common genotype frequently observed in rifampicin-resistant Mycobacterium tuberculosis clinical isolates. In summary, we provide a cell-free synthetic biology strategy for the profiling of antibiotic sensitivity and resistance from K. pneumoniae. While initial processing requires Biosafety Level 2, the final extracts are non-living and suitable for long-term storage, and potentially transfer to a Biosafety Level 1 lab. This bioassay has potential uses for early-stage host-specific antimicrobial development and the testing of AMR variants for structure-activity relationship studies. The data reposited is label-free high-resolution LC-MS proteomics data performed to characterise the proteins in cell-free extract of K. pneumoniae ATCC 13882 and compare to that of E. coli MG1655 to identify common and unique proteins. We also characterised the proteins of K. pneumoniae clinically resistant isolates ST258-T1b and NJST258-1, and compared them to K. pneumoniae ATCC 13882 laboratory strain.

2023-11-29 | PXD046861 | Pride

A cell-free strategy for profiling intracellular antibiotic sensitivity and resistance – characterising the proteomes of K. pneumoniae ATCC 13882, ST258-T1b and NJST258-1 and E. coli K-12 MG1655.

2023-11-29 | PXD046864 | Pride

Metabolic Dynamics in Escherichia coli-Based Cell-Free Systems

Project description:The field of metabolic engineering has yielded remarkable accomplishments in using cells to produce valuable molecules, and cell-free expression (CFE) systems have the potential to push the field even further. However, CFE systems still face some outstanding challenges, including endogenous metabolic activity that is poorly understood yet has a significant impact on CFE productivity. Here, we use metabolomics to characterize the temporal metabolic changes in CFE systems and their constituent components, including significant metabolic activity in central carbon and amino acid metabolism. We find that while changing the reaction starting state <i>via</i> lysate preincubation impacts protein production, it has a comparatively small impact on metabolic state. We also demonstrate that changes to lysate preparation have a larger effect on protein yield and temporal metabolic profiles, though general metabolic trends are conserved. Finally, while we improve protein production through targeted supplementation of metabolic enzymes, we show that the endogenous metabolic activity is fairly resilient to these enzymatic perturbations. Overall, this work highlights the robust nature of CFE reaction metabolism as well as the importance of understanding the complex interdependence of metabolites and proteins in CFE systems to guide optimization efforts.

2023-02-22 | MTBLS2630 | MetaboLights