Coding

Part:BBa_K2020053

Designed by: Andrea Hoeltken, Carolina Bonerath, Vroni Czotscher   Group: iGEM16_Aachen   (2016-10-15)
Revision as of 23:47, 19 October 2016 by Carolina.bn (Talk | contribs) (→‎Screening Results)


DMNBS-Synthetase for use in E.coli

This is a DMNBS-synthetase to be used as a orthogonal synthetase in E.coli. This part can be used together with the cognate tRNA BBa_K2020042 to incorporate DMNBS in response to an amber stop codon.

Compared to incorporation of tyrosine with wild type Mj Y-Synthetase in response to an amber codon:

  • Incorporation Efficiency: % (DMNBS incorporation value)
  • Incorporation Fidelity: %(discrimination againt tyrosine)

Full Set of DMNBS synthtetases

Here are some evolved synthetases from [http://2016.igem.org/Team:Aachen iGEM Team Aachen 2016], which have been evaluated with Flourescent reporter for measurement of incorporation of ncAA.

Usage and Biology

Incorporation of ncAA

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 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 21st 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.

Assembly in a synthetase plasmid for incorporation of ncAA

alt text

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.

Host organism

This synthetase plasmid and the corresponding measurement of protein formation is previously used in BL21 DE3 gold resulting in competion of the supressor tRNA with release factor one at the amber stop codon at the usual 321 amber stop codons. Other tRNA/synthetase pairs working according to amber codon supression have also been used in an amberless E.coli strain.

Screening Results

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. 2. This mutant corresponds to No.: 2

Fig 2: Efficiency (dark green) and fidelity (light green) of various DMNBS mutants compared to wild type Mj tyrosyl synthetase

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 84
    Illegal SapI.rc site found at 877


[edit]
Categories
Parameters
None