Download In vitro translation with non

PhD project
In vitro translation with non-natural amino acids
Drs. Hine, Collighan and Spickett
Project Summary:
This project aims to incorporate unnatural amino acids (UAA’s) into proteins via in vitro translation and will be
delivered via a series of work packages as follows:
WP1: Establishing in vitro translation within the Hine/Collighan labs
Objective 1.1. In vitro synthesis of tRNA. The GTA will work with Dr M Ashraf to learn how to produce
synthetic tRNA via run-off transcription. A set of 20 native tRNAs will then be produced. A 21st
tRNA that does not cross-react with codons bound by the 20 native tRNAs will also be produced
for in vitro charging.
Objective 1.2. Establishing conventional in vitro translation. Using a commercial system (NEB PURExpress,
the GTA will work with Drs Collighan & Hine to develop the skills to use commercial in vitro
translation kits (NEB Pure), producing native luciferase from in vitro transcription of a luciferase
plasmid.
Objective 1.3. Synthetic tRNA charging. Working with Dr Hine, the student will follow literature precedent
(eg. Chem. Commun., 2005, 4321–4323 and refs therein) to charge the 21st synthetic tRNA
(objective 1.1.) with a fluorescently-labelled unnatural amino acid.
Objective 1.4. Establishing in vitro translation with UAAs. A synthetic luciferase gene will be purchased that
uses only one codon per native aa, with a 21st codon only in the active site. The set of 20 native
tRNAs (objective 1.1.) and the synthetically-charged tRNA will be added to the PURExpress
(aa, tRNA) kit, along with the kit’s native aa mixture and the synthetically-charged 21st tRNA.
The mixture will be used to translate the synthetic gene and the protein product examined via
the ultra-sensitive CCD camera in the Collighan lab. The process will be optimized to maximize
UAA incorporation.
WP2: Development of non-natural mutagenesis of luciferase within the Hine/Collighan labs
Objective 2.1. Production of multiple UAA/tRNAs. Experience generated in WP1 will be applied to generate
multiple synthetic tRNA/UAA combinations.
Objective 2.2. Extended mutagenesis of luciferase. Again, experience generated in WP1 will be used to
generate various single and multiple non-natural substitutions within the active site of
luciferase. These experiments will be informed by the extensive experience of the Collighan lab
in luciferase mutagenesis (codons in the synthetic gene will be mutated via Quik-change or
ProxiMAX mutagenesis as required).
Objective 2.3. Analysis of mutants. The various “mutant” proteins will be examined for altered activity in the
Collighan lab.
WP3: Development of oxidized proteins for analysis within the Spickett lab
Objective 2.4. Production of tRNAs bearing oxidized aa’s. Following from objectives 1.3, 1.4 and 2.1, tRNAs
bearing commercially-available, oxidized aa’s will be produced.
Objective 2.5. In vitro translation and analysis of oxidized proteins. Experience generated in WP1/WP2 will
be used to generate various single and multiple oxidized aa substitutions within proteins
relevant to MASSTRPLAN (an ITN involving multiple, international researchers, co-ordinated by
Dr Spickett). Analysis of the resulting proteins will involve co-ordination with MASSTRPLAN
researchers in the Spickett lab. The project will generate novel technology to produce modified
proteins and study their effects in vitro and in vivo.
PhD project
In vitro translation with non-natural amino acids
Drs. Hine, Collighan and Spickett
Ability of the Supervisory team to deliver the project:
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The Hine research group is experienced in producing synthetic tRNA via in vitro run-off transcription
and is also currently mutating the amino acid binding site of E. coli alanyl tRNA synthetase (in
collaboration with Isogenica Ltd) via ProxiMAX randomization, to bind multiple different aa’s. AlaRS
already has the ability to bind multiple tRNAs, since it discriminates only via the acceptor arm of tRNA,
rather than the anticodon. Having previously developed ProxiMAX randomization, which uses only one
codon for each amino acid, these latest individual projects form part of a larger goal which aims to
develop ProxiMAX randomization to translate with up to 12 unnatural amino acids (UAA’s) in addition
to the 20 natural aa’s (total 32 aa’s). The GTA will contribute to that greater goal, via individual subprojects with Drs Collighan and Spickett.
The Collighan research group has extensive experience of working with firefly luciferase. They have
generated mutations that improve thermostability or detergent stability, alter kinetics and change
emitted light colour. With the exquisite sensitivity that light detection offers, firefly luciferase can be
assayed at extremely low concentrations, making it ideal for production by in vitro translation systems.
This project will allow rapid and efficient screening of the effects of single/multiple substitutions at
chosen positions. Extension of the repertoire of 20 natural amino acids by UAAs will allow more scope
for the production of completely novel mutants, potentially with altered functionality and will provide
more insight into the mechanism involved.
The Spickett research group has extensive experience of studying the effects of oxidized amino acids
within proteins and will provide exposure for the GTA to international researchers, via the
MASSTRPLAN project.
All supervisors have extensive experience of supervising PhDs, with 100% record of submission within 4
years (students progressing beyond year 1) and all resulting graduates achieving postdoctoral-level
employment.