Summary of study ST000446

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000344. The data can be accessed directly via it's Project DOI: 10.21228/M8KK69 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST000446
Study TitleHeterologous expression and detection of Apratoxins in E. coli
Study TypeOrganic extraction of E coli cultures harboring apratoxin gene cluster
Study SummaryThe apratoxin gene cluster was recovered from fosmid DNA library. The gene set responsible for the biosynthesis of the polyketide backbone was introduced in E. coli BAP strain and expressed at 30C for 24 hours. Two sets of control and experimental samples with culture broth pH adjusted at 8 and 12 respectively was extracted with ethyl acetate and dried.
Institute
University of Florida
DepartmentSECIM
Last NameKallifidas
First NameDimitris
AddressMedical Sciences Building, Rm P5-26, 1600 SW Archer Rd., FL32610
Emaildkallifidas@ufl.edu
PhoneNone
Submit Date2016-07-28
Num Groups1
Total Subjects12
Analysis Type DetailLC-MS
Release Date2017-10-03
Release Version1
Dimitris Kallifidas Dimitris Kallifidas
https://dx.doi.org/10.21228/M8KK69
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:


Project:

Project ID:PR000344
Project DOI:doi: 10.21228/M8KK69
Project Title:Cloning, expression and derivatization of cytotoxic marine natural product Apratoxin
Project Summary:Apratoxin is a hybrid PKS/NRPS secondary metabolite showing potent anticancer activity by inducing G1 phase cell cycle arrest and apoptosis. There have been reported a series of natural apratoxin derivatives with defined chemical modifications in their structures that alter their biological activity. Looking for a better activity, total synthesis of apratoxin has been achieved and a series of synthetic congeners showed increased activity with subnanomolar potency. From the total synthesis point of view, the modification of the polyketide backbone presents a burden for a radical derivatization of apratoxins. We hypothesized that supply the polyketide part naturally by overexpression the corresponding genes it will facilitate the generation of apratoxin congeners by decorating the backbone with a variety of amino acids improving the antitumor activity.Threfore, the apratoxin gene cluster was recovered from a fosmid DNA library. The gene set responsible for the biosynthesis of the polyketide backbone was introduced in E. coli BAP strain and expressed at 30C for 24 hours. Two sets of control and experimental samples with culture broth pH adjusted at 8 and 12 respectively was extracted with ethyl acetate and dried.
Institute:University of Florida
Department:Medicinal Chemistry
Last Name:Luesch
First Name:Hendrik
Address:Medical Sciences Building, Rm P5-26, 1600 SW Archer Rd., FL32610
Email:luesch@cop.ufl.edu
Phone:none

Subject:

Subject ID:SU000467
Subject Type:bacteria cells
Subject Species:Escherichia coli
Taxonomy ID:562
Species Group:Microorganism

Factors:

Subject type: bacteria cells; Subject species: Escherichia coli (Factor headings shown in green)

mb_sample_id local_sample_id pH treatment
SA022652E.coli+bAprat14_pH12_212 bAprat14
SA022653E.coli+bAprat14_pH12_312 bAprat14
SA022654E.coli+bAprat14_pH1212 bAprat14
SA022655E.coli_control_pH1212 control
SA022656E.coli_control_pH12_212 control
SA022657E.coli_control_pH12_312 control
SA022658E.coli+bAprat14_pH8_38 bAprat14
SA022659E.coli+bAprat14_pH8_28 bAprat14
SA022660E.coli+bAprat14_pH88 bAprat14
SA022661E.coli_control_pH8_28 control
SA022662E.coli_control_pH88 control
SA022663E.coli_control_pH8_38 control
Showing results 1 to 12 of 12

Collection:

Collection ID:CO000461
Collection Summary:50 ml of LB broth inoculated with the appropriate E. coli strains were extracted twice with equal volume of etheyl acetate and dried.
Sample Type:Dried ethyl acetate cell extracts

Treatment:

Treatment ID:TR000481
Treatment Summary:The set of genes responsible for the biosynthesis of the Apratoxin polyketide backbone was introduced in E. coli BAP strain and expressed at 30C for 24 hours. Two sets of control and experimental samples with culture broth pH adjusted at 8 and 12 respectively was extracted with ethyl acetate and dried.
Cell Media:Luria Bertani (LB) broth

Sample Preparation:

Sampleprep ID:SP000474
Sampleprep Summary:none
Sampleprep Protocol Filename:Consolidated_Cellular_Metabolomics_SOP.pdf
Sample Spiking:Appendix A - Internal Standard Prep GLCMS.pdf

Combined analysis:

Analysis ID AN000697 AN000698
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Scientific-Dionex Ultimate 3000 Thermo Scientific-Dionex Ultimate 3000
Column ACE Excel 2 C18-PFP (100 x 2.1mm, 2um) ACE Excel 2 C18-PFP (100 x 2.1mm, 2um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE
Units peak area peak area

Chromatography:

Chromatography ID:CH000505
Methods Filename:Metabolomics_LCMSProtocol.pdf
Instrument Name:Thermo Scientific-Dionex Ultimate 3000
Column Name:ACE Excel 2 C18-PFP (100 x 2.1mm, 2um)
Chromatography Type:Reversed phase

MS:

MS ID:MS000619
Analysis ID:AN000697
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:HESI
Ion Mode:POSITIVE
  
MS ID:MS000620
Analysis ID:AN000698
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:HESI
Ion Mode:NEGATIVE
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