Part:BBa_K1416004:Experience

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Applications of BBa_K1416004

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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 gained experience in cultivation and created some data, which may be helpful to other users. Addionally we created a new part ( Flourescent reporter for measurement of incorporation of ncAA ), to make the screening system available through the registry of standard biological parts.

Documentation of the improvement

How the reporter plasmid works

Fig 1:Reporter Plasmid

This reporter plasmid is one of a two plasmids containing screening system for determining efficiancy and fidelty of non-canonical amino acids (ncAA) incorporation via amber termination supression. One plasmid contains tRNA and corresponding aminoacylation-synthetase. The other one is this plasmid presented herein. It consists of am mRFP1 domain which is connected through a linker sequence containing a recoded amber stop codon with a sfGFP domain. The expression of the plasmid gives either a red fluorescence, or - if the ncAA will be incorporated at the recoded amber stop codon within the linker site - both a red and a green fluorescence.

Synthetase and tRNA are constitutivly expressed in a plasmid with low copy replicon p15A, taking into account that expression results in metabolic stress, but are not under IPTG control for he purpose of avoiding abrupt and unpredictable effects considering extra time and energy needed for their assembly. Whereas the reporter plasmid containig two fluorescence proteins, is kept under operon control for IPTG induction likewise on a low copy plasmid with ColE1. [picture reporter plasmid]

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.

Host organism

This reporter plasmid and the corresponding measurement of protein formation is previously used in both an amberless E.coli strain and BL21 DE3 gold. The use of the latter is resulting in competion of the supressor tRNA with release factor one at the amber stop codon at the usual 321 amber stop codons.

Measurement: Wavelength

Fig 2: normalized fluorescence spectrum of mRFP1

Fig 3: 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 2, Fig 3). 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: Example of Evaluation

A first approximation of efficiency and fidelity can be made by normalizing GFP levels of the synthetase to be evaluated to a well working synthetase if the levels of optical density are equal. Thus you eliminate the biogenic background fluorescence levels and compare the clones to each other. Refer Fig.4.

Fig 4: Effieciency (dark green) and fidelity (light green) of various DMNBS mutants compared to wild type Mj tyrosyl synthetase

With the reporter plasmid evaluated synthtases

Links

Part K2020040 - Flourescent reporter for measurement of incorporation of ncAA : Whereas you find hereunder the whole plasmid as a biobrick, we provide the two flourescent proteins connected through the linker sequence as a biobrick for self-assembly in a low copy plasmid.
[http://2014.igem.org/Team:Austin_Texas/kit Team Austin, Texas 2014 built this measurement kit and probed various synthetases]
[http://2016.igem.org/Team:Aachen Team Aachen 2016 evolved a new synthetase for incorporation of DMNBS in E.coli]

Attributions

iGEM Team Austin, Texas: Thanks for making the measurement kit available to us.

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

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