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

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

It was shown previously, that highest GFP formation is achieved with supplementation of IPTG and the ncAA at the beginning of incubation, but does results in decelerated growth (1). In fact, cultivation of BL21 DE3 gold containing two different plasmids show lower growth rates, when adding 100µM IPTG and 2mM DMNBS to M9 minimal medium, resulting overall in a growth phase of 42-48h at 30°C to reach maximum cell density.

Measurement: Wavelength

normalized fluorescence spectrum of mRFP1

normalized fluorescence spectrum of sfGFP

Measurement: Evaluation

Links