Team:DTU-Denmark/Project/Overview


Overview


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Project

Introduction

Over the summer we worked with nonribosomal peptide synthetases (NRPSs). We developed a B. subtilis strain capable of oligo-mediated genome engineering and used this strain to alter a NRPS. We also investigated methods of screening for novel products with desired activities.

Background

Nonribosomal peptide synthases (NRPSs) are large multimodular enzymes that synthesize nonribosomal peptides, which are short bioactive peptides with a broad range of functions, including antibiotics, immunosuppressants and anticancer drugs.

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MAGE subtilis

Multiplex automated genome engineering (MAGE) utilises cyclical recombination with short oligonucleotides in order to achieve a high allelic replacement efficiency and can be used to quickly generate cell populations with varying phenotypes. We introduced oligo-mediated genome engineering into Bacillus subtilis and used it to alter a nonribosomal peptide synthetase.

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Tyrocidine

Tyrocidine is a mixture of non-ribosomal peptides. It can only be used topically due to its toxicity. We sought to express the tyrocidine synthase cluster in B. subtilis to make novel derivatives with oligo-mediated recombineering.

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Lab-on-a-disc

Lab-on-a-disc is a potential screening method for our MAGE NRPS products. After screening for antimicrobial activity, prospective MAGE edited NRPS products could be tested for different beneficial properties. One example is a quick screening method for cytotoxicity.

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Intein

When one method fails the Synthesizer team has to innovate! We explored an alternative approach for generating short, cyclized peptides with similar length to tyrocidine by using self-splicing proteins. These short, self-splicing proteins that have no function in the proteins they are a part of, besides catalyzing their own excision after translation, are called inteins. The splicing creates a peptide bond between the two adjacent amino acids next to the inteins and we hypothesized that it could be used to make our cyclic peptides.

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NRP Detection

Separation and identification of our non-ribosomal peptide synthetase (NRPS) products was determined by ultra-high performance liquid chromatography with diode array detection coupled to quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QTOFMS). NRPS variants were identified by changes in mass-to-charge ratio and column retention time. 

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Technical University of Denmark
Department of Systems Biology
Søltofts Plads 221
2800 Kgs. Lyngby
Denmark
P: +45 45 25 25 25
M: dtu-igem-2015@googlegroups.com