Actinomadura Species: Laboratory Maintenance and Ribosome Engineering

Dipesh Dhakal1, Nguyen Thanh Chung1, Vijay Rayamajhi1, Jae Kyung Sohng2

1 Department of Life Science and Biochemical Engineering, Sun Moon University, Asan‐si, 2 Department of BT‐Convergent Pharmaceutical Engineering, Sun Moon University, Asan‐si
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 10G.1
DOI:  10.1002/cpmc.22
Online Posting Date:  February, 2017
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Abstract

Actinomadura spp. are aerobic, Gram‐positive, catalase‐positive, non‐acid fast, non‐motile actinomycetes. Some species of Actinomadura are associated with opportunistic infections in humans. However, many bioactive compounds with pharmaceutical applications can be isolated from various Actinomadura spp. This unit includes general protocols for the laboratory maintenance of Actinomadura spp., including growth in liquid medium, growth on solid agar, long‐term storage, and generation of a higher producing strain by ribosome engineering. Actinomadura hibisca P157‐2 is used as a prototype for explaining the considerations for efficient laboratory maintenance of Actinomadura spp. © 2017 by John Wiley & Sons, Inc.

Keywords: Actinomadura spp; growth medium; storage; ribosome engineering

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Growth of Actinomadura on Solid Medium
  • Basic Protocol 2: Growth of Actinomadura in Liquid Medium
  • Basic Protocol 3: Long‐Term Storage of Actinomadura
  • Basic Protocol 4: Production of Secondary Metabolites (Pradimicins) from Actinomadura Hibisca P157‐2
  • Basic Protocol 5: Generation of Higher Secondary Metabolite (Pradimicins) Producing Strain of Actinomadura by Ribosome Engineering
  • Reagents and Solutions
  • Commentary
  • Literatures Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Growth of Actinomadura on Solid Medium

  Materials
  • ISP2 or ISP4 medium (see recipe) with appropriate antibiotics
  • Actinomadura hibisca P157‐2 glycerol stock (see protocol 3) or frozen stock (ATCC #53557)
  • 100 × 15‐mm petri dishes
  • Sterile inoculation needle or wooden stick
  • 28°C incubator

Basic Protocol 2: Growth of Actinomadura in Liquid Medium

  Materials
  • ISP2 liquid medium (see recipe) with appropriate antibiotics
  • Actinomadura hibisca P157‐2 on solid medium ( protocol 1) or glycerol stock ( protocol 3)
  • 250‐ml baffled culture flask, sterile (glass or plastic with vented caps)
  • Toothpicks, sterile
  • Sterile cotton
  • 28°C shaking incubator

Basic Protocol 3: Long‐Term Storage of Actinomadura

  Materials
  • Actinomadura hibisca P157‐2 liquid culture (see protocol 2)
  • Sterile glycerol
  • 1.5‐ml microcentrifuge tube
  • −80°C freezer

Basic Protocol 4: Production of Secondary Metabolites (Pradimicins) from Actinomadura Hibisca P157‐2

  Materials
  • Regenerated seed of Actinomadura hibisca P157‐2 in ISP2 liquid medium (see protocol 2)
  • GYSP medium (see recipe)
  • 6 N HCl
  • Ethyl acetate
  • n‐Butanol
  • Methanol
  • 500‐ml baffled culture flask, sterile (glass or plastic with vented caps)
  • 28°C shaking incubator
  • Centrifuge
  • Separatory funnel
  • Rotary evaporator

Basic Protocol 5: Generation of Higher Secondary Metabolite (Pradimicins) Producing Strain of Actinomadura by Ribosome Engineering

  Materials
  • Regenerated seed of Actinomadura hibisca P157‐2 in ISP2 liquid medium (see protocol 2)
  • ISP2 agar plates supplemented with streptomycin (see recipe)
  • ISP2 liquid medium (see recipe) with and without appropriate concentrations of streptomycin
  • 500‐ml baffled culture flask, sterile (glass or plastic with vented caps)
  • Sterile cell spreader
  • 28°C incubator
  • Additional reagents and equipment for long‐term storage ( protocol 3) and production of secondary metabolites ( protocol 4)
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Figures

Videos

Literature Cited

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Internet Resources
   http://www.bacterio.net/index.html
  Valuable resource for anyone with an interest in prokaryotic nomenclature.
   http://www.bacterio.net/actinomadura.html
  Comprehensive information about different Actinomadura species.
   http://www.cdc.gov/biosafety/publications/bmbl5/index.htm
  Guidelines about the critical considerations for biosafety in microbiological and biomedical laboratories.
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