Part:BBa_K4425000

Trp repressor

Trp repressor adopt a conformation change that amino acid 66-86 into a stable HTH motif which allow the repressor to interact with the operator with high affinity. Downstream gene expression is suppressed when trp repressor bind operator.

Usage and Biology

This is a mutant that is mutated by a wild-type tryptophan repressor that can bind to mutant trp operator mutant O1 (GATGTAGTTAACTACAACGCA) with high affinity. Both trp and Br-trp are valid ligand to the trp repressor with a little difference in affinity (Fig. 1).

Figure 1: the trp repressor was tested in the presense of trp or Br-trp (1mM), and maxmal promoter expression was measured

Source

generate by Ellefson et al, 2018

Characterization

We construct pTrpR to express trp repressor and co-transform pTrpR with pNEG into DH5α. We tested the RPU/OD600 which stand for promoter expression of the induced cell, under the condition of different concentration of Trp. However, it can only provide about three times the inhibition effect in promoter expression.

Figure 2: schemtic of pTrpR and pNEG

Figure 3: GFP production (RPU/OD600) – trp concentration graph by GFP test for wild type trp repressor and pNEG.

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 315
  

Reference

Ellefson, J. W., Ledbetter, M. P., & Ellington, A. D. Directed evolution of a synthetic phylogeny of programmable Trp repressors. Nature chemical biology, 2018, 14(4), 361–367.