Part:BBa_K3645011

Target-AID (CBE)

Contains the full CDS of Target-AID, whose Cas9 part was replace with our lab's dCas9.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1099
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4775
  Illegal BamHI site found at 3378
  Illegal XhoI site found at 4384
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Contribution From NNU-China 2021

Group: iGEM Team NNU-China 2021

Author: Yan Xu

Summary: Testing its gene editing efficiency in BL21 (DE3)

Characterization from iGEM21-NNU-China

        Microbially produced 2‑Phenylethanol (2-PE) is mainly obtained by two routes, containing the de novo pathway from glucose and bioconversion from L-phenylalanine by the Ehrlich pathway. Compared to the multi-step pathway, the Ehrlich pathway provides more efficient synthesis of 2-PE In the Ehrlich pathway, the generation of L-phenylalanine by oxidative decarboxylation to phenylethylaldehyde is a key step in the high yield of 2-PE (Farhi et al. 2010) (Fig. 1). Petunia hybrida hybrid phenylacetaldehyde synthase (PAAS) was first registered in 2021, which had been shown to have functional activity in Saccharomyces cerevisiae BY4741. We introduced this gene into the genome of Y. lipolytica polf and tested it in shake flask fermentation with 4 g/L of L-phe. The experimental result showed that overexpression of PhPAAS (BBa_K4297067) does not affect the normal growth of Y. lipolytica. Simultaneously, there was a significant increase in the yield of 2-PE, reaching 957.35 mg/L (Fig. 1). These results provide references for future iGEM team to select suitable sources of PAAS.

Fig. 1. A. The key synthetic steps of the Ehrlich pathway. B. Overexpression of PhPAAS enhances 2-PE yield.

Reference

1. Farhi M, Lavie O, Masci T, Hendel-Rahmanim K, Weiss D, Abeliovich H, Vainstein A (2010) Identification of rose phenylacetaldehyde synthase by functional complementation in yeast. Plant Mol Biol 72(3):235-245.