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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
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.
| Study ID | ST000446 |
| Study Title | Heterologous expression and detection of Apratoxins in E. coli |
| Study Type | Organic extraction of E coli cultures harboring apratoxin gene cluster |
| Study Summary | The 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 |
| Department | SECIM |
| Last Name | Kallifidas |
| First Name | Dimitris |
| Address | Medical Sciences Building, Rm P5-26, 1600 SW Archer Rd., FL32610 |
| dkallifidas@ufl.edu | |
| Phone | None |
| Submit Date | 2016-07-28 |
| Num Groups | 1 |
| Total Subjects | 12 |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2017-10-03 |
| Release Version | 1 |
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: | Bacteria |
Factors:
Subject type: bacteria cells; Subject species: Escherichia coli (Factor headings shown in green)
| mb_sample_id | local_sample_id | pH | treatment |
|---|---|---|---|
| SA022652 | E.coli+bAprat14_pH12_2 | 12 | bAprat14 |
| SA022653 | E.coli+bAprat14_pH12_3 | 12 | bAprat14 |
| SA022654 | E.coli+bAprat14_pH12 | 12 | bAprat14 |
| SA022655 | E.coli_control_pH12 | 12 | control |
| SA022656 | E.coli_control_pH12_2 | 12 | control |
| SA022657 | E.coli_control_pH12_3 | 12 | control |
| SA022658 | E.coli+bAprat14_pH8_3 | 8 | bAprat14 |
| SA022659 | E.coli+bAprat14_pH8_2 | 8 | bAprat14 |
| SA022660 | E.coli+bAprat14_pH8 | 8 | bAprat14 |
| SA022661 | E.coli_control_pH8_2 | 8 | control |
| SA022662 | E.coli_control_pH8 | 8 | control |
| SA022663 | E.coli_control_pH8_3 | 8 | 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) |
| Flow Rate: | 0.350mL/min-0.600mL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile |
| 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 |
| Analysis Protocol File: | Metabolomics_LCMSProtocol.pdf |
| MS ID: | MS000620 |
| Analysis ID: | AN000698 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | HESI |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | Metabolomics_LCMSProtocol.pdf |
