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 works 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

Fig 1: normalized fluorescence spectrum of mRFP1

Fig 2: normalized fluorescence spectrum of sfGFP

As a prescreening setup an endpoint detection of OD and fluorescent intensities with Tecan Plate Reader is chosen. Excitation and emission wavelegthes of mRFP1 and sfGFP were obtained from a previously conducted measurement with a Biolector (Fig 1, Fig 2). For screening with Tecan Plate Reader the following settings were used:

• OD: 600 nm
• sfGFP 480/508 nm
• mRFP1 584/605 nm
• Slid: 8 nm
• 30°C
• Shaking in between measurements

For rescreening the same plate reader setup for endpoint detection is used. Additionally an online measurement monitoring sfGFP and mRFP1 formation as well as scattered light at 650nm is used. This is achieved by a screening platform constructed inhouse based on the established Biolector setup. A quartz/quartz multi-mode fibre is moved sequentially below the wells of the microtiter plates and is connected to a spectrophotometer with extinction/emission monochromators (1). This allows quasi-continous and contactless measurements while shaking.

Measurement: Evaluation

Links

References

1. Wandrey et al, 2016, Probing unnatural amino acid integration into enhanced green fluorescent protein by genetic code expansion with a high-throughput screening platform