Difference between revisions of "Part:BBa K2020050"

Revision as of 13:39, 17 October 2016

wild type Mj Y-Synthetase for use in E.coli

This is the wild type tyrosyl synthetase derived from Methanococcus janaschii to be used as a orthogonal synthetase in E.coli. This part can be used together with the cognate tRNA BBa_K2020042 to incorporate Tyrosin in response to an amber stop codon.

Usage and Biology

Incorporation of ncAA with amber codon supression

Photocleavable non-canonical amino acids offer the opportunity to control protein function on a non-invasive basis. Working with unnatural amino acids requires an additional, orthogonal pair of a tRNA and a cognate synthetase i.e. which does not crossreact with the endogenous tRNA/synthetase pairs [1]. The tRNA's anticodon contains is mutated to amber stop anticodon. Hence, it is possible to incorporate an amino acid at a chosen position in a protein via amber codon suppression.

A previously reported tRNA/synthetase pair for O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine (DMNBS) which derived from Escherichia coli and was used in Saccharomyces cerevisiae [1] leads to the lack of a possibility to work with non-canonical amino acids replacing serine in E. coli by using a 21^st amino acid.

Based on computational modeling a synthetase for DMNBS in E. coli is designed by Team Aachen 2016 with by creating a semi rational mutation library. We made the clones with highest incorporation effiency and fidelity available for further research.

Control for incorporation of ncAA

When working with amber codon supression the Methanococcus wild type tyrosyl synthetase is a suitable control, as tyrosine is present even in minimal media.

Assembly in a synthetase plasmid for incorporation of (n)cAA

pACYC derived plasmid with Mj tyrosyl synthetase and cognate tRNA

Most synthetases are used with low copy plasmids (e.g. pACYC). Assemble the tRNA and the synthetase into a low copy plasmid, each one with an own promoter and one terminator for both. (See picture). If your application is not for incorporation into a protein but for use with a second plasmid, make shure to use replicons from different incompatibility groups, eg. ColE1 and p15A and different selection markers. A second plasmid could be the flourescent reporter plasmid pFRY for the purpose of determining fidelity and efficiacy of synthetases for ncAA.

Recognition between tRNA and (n)cAA-synthetases derived from Mj

Methanococcus janaschii wild type tyrosyl tRNA consists of two arms: Firstly the acceptor-minihelix, where the amino acid will be attached to the 3' end. Secondly the anticodon containg arm. Synthetases interact mainly with the acceptor minihelix of the tRNA. Due to the lack of most of a recognizing element within the anticodon containg section, a mutation of a anticodon base has a relatively small effect on the aminoacylation efficiency [2] and may explain why a variety of ncAA can be incorporated with this tRNA.

Example measurement proving incorporation of amino acids

Fig 2: normalized fluorescence spectrum of mRFP1

Fig 3: normalized fluorescence spectrum of sfGFP as a sign for successful amino acid incorporation via tRNA/synthetase pair for tyrosine via amber supression

Wild Type Methanococcus janaschii tRNA/synthetase pair for incorporation of tyrosine at an amber termination codon is cotransformed with pFRY - Flourescent reporter plasmid for measurement of incorporation of ncAA into BL21 DE3 gold. Reporter plasmid is induced by 100µM ITPG.

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

This experiment is performed in order to obtain fluorescence spectra of mRFP1 and sfGFP. As you can see in Fig.3 GFP formation was measured as a result from successful amino acid incorporation via amber supression.

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

Sequence and Features