Team:Heidelberg/Project/Indigoidine-Tag

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Revision as of 19:45, 25 October 2013 by Philipp.walch (Talk | contribs)

Indigoidine-Tag. Inventing the GFP for NRPs.

Highlights

  • Creation of an easily detectable NRPS-Tag using the Indigoidine Synthetase
  • Proof of principle for inter-species module shuffling
  • Protocol for High-Throughput NRPS-assays: RFC 100 and RFC 99
  • Empirical determination of optimal domain boarders for T-domain exchange
  • Alteration of enzyme activity by exchange of T-domain
  • Proof of functionality for synthetic T-domains

Abstract

An integral characteristic of synthetic biology yet often undermined is the ability to learn fundamental knowledge by systematically perturbing a biological system. Non-ribosomal peptide synthetases (NRPS) are predestinated for such a trial and error approach. Their hierarchical organization into modules and domains offer a unique opportunity to spin around their inherent logical assembly and observe if their functionality is preserved. Following this idea, interchangeability of modules within one pathway has already been proven by both, us (LINK) and several research groups (LINK). However, detection of the product, as well as high-throughput screening of functionality remained complicated. Indigoidine, a blue pigment which is synthesized by the NRPS IndC from Photorhabdus luminescens laumondii TT01 (DSM15139), was established as a novel and application-oriented tag functionally fused to synthesized peptides. Streamlining this process demonstrates a substantial reinforcement of the NRPS-Designer, our software tool, and offers the possibility to evaluate and optimize synthesis of Non-Ribosomal Peptides (NRPs) in a high-throughput manner (RFC 99). Furthermore, we established an Indigoidine-production assay based on OD measurement of the blue-colored pigment. We thereby could prove the dependence of the efficiency on the T-domain and the 4'-Phosphopanthetheinyl-transferases (PPTases), resulting in different levels of Indigoidine synthesis.

Introduction

Start introduction here ...

Results

Result 1

Result 1.1

Result 1.2

Discussion

Lets discuss ...


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Thanks to

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