Part:BBa_K2020052

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 iGEM Team Aachen 2016, which have been evaluated with Flourescent reporter for measurement of incorporation of ncAA.

• 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

Usage and Biology

Assembly in a synthetase plasmid for incorporation of ncAA

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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.

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]

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