Difference between revisions of "Part:BBa K2020040"

Revision as of 02:35, 3 December 2016

Fluorescent reporter for measurement of incorporation of ncAA

pRXG is one part of a two plasmid reporter system for measurement of incorporation of ncAA via amber supression. It consists of two fluorescent domains connected by a linker sequence containing and amber stop. When IPTG induced and expressed, the fluorescence intensity can be measured. A red fluorescence is always visible upon induction, and if an amino acid is incorporated as response to the amber stop codon, a green fluorescence intensity is measurable. Fidelty and efficiencycy of the incorporation can be determined with comparison of fluorescent level.

Usage and Biology

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 constitutively expressed in a plasmid with low copy replicon, taking into account that expression results in metabolic stress, but are not under IPTG control for the 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]

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.

Assembly in a plasmid

Most synthetases are used with low copy plasmids (e.g. pACYC). The reporter should also be used with a low copy replicon. Assemble the part provided here into a low copy plasmid, make sure to use replicons from different incompatibility groups, eg. ColE1 and p15A and different selection markers. Keep under operon control for induction.

Example of cultivation conditions with High Throughput measurement

Example taken from [http://2016.igem.org/Team:Aachen|Team Aachen 2016 - Evolving a new synthetase for DMNBS incorporation in E.coli]

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 competent cells containing already 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 2: normalized fluorescence spectrum of mRFP1

Fig 3: normalized fluorescence spectrum of sfGFP

Excitation and emission wavelegthes of mRFP1 and sfGFP were obtained from a conducted measurement with a Biolector set up (1)(Fig 2, Fig 3).

For screening with a plate Reader the following settings can be used:

• OD: 600 nm
• sfGFP 480/508 nm
• mRFP1 584/605 nm
• Slid: 8 nm

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

• Y-RS, canonical amino acid
• oNBY-RS
• AzF-synthetase
• CN-F synthetase
• Iodo-Y synthetase
• 5HT-P synthetase
• Nitro-Y synthetase
• Amino-Y synthetase

DMNBS-RS clones:

• • DMNBS-RS Clone 1
  • DMNBS-RS Clone 2
  • DMNBS-RS Clone 3
  • DMNBS-RS Clone 4
  • DMNBS-RS Clone 5
  • DMNBS-RS Clone 6
  • DMNBS-RS Clone 7
  • DMNBS-RS Clone 8
  • DMNBS-RS Clone 9
  • DMNBS-RS Clone 10
• OMe-Y-RS

Links

• Part K1416004: Improved original part provided by iGEM Austin, Texas 2014
• [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]

Sequence and Features