Difference between revisions of "Part:BBa K1416004:Experience"

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===Documentation of the improvement===
 
===Documentation of the improvement===
  
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====Cultivation conditions====
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====Cultivation conditions with High Throughput measurement====
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In order to evolve a new aminoacylation synthetase for DMNBS in E.coli, transforming a mutation library into competent cells the following order worked to get a maximum output and equal optical densities:
 +
 
 +
 
 +
*<b>Transform</b> into (BL21 DE3 gold + pFRY) on M9 solid, growth: 2 days, 37°C
 +
*<b>Pick</b> into M9 liquid: <b>Masterplate</b>, growth: 2 days at 30°C, 900rpm,  shaking diameter: 50 mm
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*<b>Replicate</b> into M9 liquid, growth 2 days at 30°C, 900 rpm, shaking diameter: 50 mm
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*<b>Replicate</b> into M9 liquid <b>Screening plate</b>, growth 2 days at 30°C, 900 rpm, shaking diameter:50 mm
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**For positive screening, supplementation at the beginning:
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***Induction with 100 µM IPTG
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***2mM DMNBS as final concentration
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**For negative screening, supplementation at the beginning:
 +
***Induction with 100 µM IPTG
  
 
====Measurement: Wavelength====
 
====Measurement: Wavelength====

Revision as of 09:14, 17 October 2016


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1416004

User Reviews

UNIQf36bae6042797682-partinfo-00000000-QINU

No review score entered. Aachen 2016, Authors: C.Bonerath, A. Hoeltken, V.Czotscher

Summary

As working with the fluorescence based screening system established by Team Austin, Texas, we created some data, which may be helpful to other users. Addionally we created a new part, to make the screening system available through the registry of standard biological parts.

Documentation of the improvement

Cultivation conditions with High Throughput measurement

In order to evolve a new aminoacylation synthetase for DMNBS in E.coli, transforming a mutation library into competent cells the following order worked to get a maximum output and equal optical densities:


  • Transform into (BL21 DE3 gold + pFRY) on M9 solid, growth: 2 days, 37°C
  • Pick into M9 liquid: Masterplate, growth: 2 days at 30°C, 900rpm, shaking diameter: 50 mm
  • Replicate into M9 liquid, growth 2 days at 30°C, 900 rpm, shaking diameter: 50 mm
  • Replicate into M9 liquid Screening plate, growth 2 days at 30°C, 900 rpm, shaking diameter:50 mm
    • For positive screening, supplementation at the beginning:
      • Induction with 100 µM IPTG
      • 2mM DMNBS as final concentration
    • For negative screening, supplementation at the beginning:
      • Induction with 100 µM IPTG

Measurement: Wavelength

normalized fluorescence spectrum of mRFP1
normalized fluorescence spectrum of sfGFP

Measurement: Evaluation

Links

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UNIQf36bae6042797682-partinfo-00000002-QINU