Part:BBa_K3753000

PAAS-Petunia

Petunia hybrida hybrid phenylacetaldehyde synthase (PAAS) is capable of transforming L-phenylalanine (L-phe) into phenylacetaldehyde by oxidative decarboxylation. Subsequently, phenylacetaldehyde can be reducted into phenylethanol, which possesses elegant, fascinating and long-lasting fragrance.

Characterization

Fig.1 The growth curve of recombinant strains.

The figure above shows the changes of biomass concentration in WT, BY4741-pRS426-Petunia, BY4741-pRS426-Vanda, BY4741-pRS426-Rosa yeast over time. The OD₆₀₀ of different strain cultures is measured at the designated time points (0h, 24h,48h,72h). The result shows that the growth trend of the recombinant strain is basically the same as the wild-type strain, indicating that the introduction of heterogeneous gene has no significant effect on the growth of yeast.

Fig.2 The 2-PE production of the recombinant strains.
After 72 hours of fermentation, 2-PE production of the wild-type of BY4741, pRS426-Petunia, pRS426-Vanda and pRS426-Rosa was tested by HPLC. Data represent means of triplicate culture±standard error.

Saccharomyces cerevisiae BY4741 contains Ehrlich pathway and other metabolic pathways to operate simultaneously to produce 2-PE, so the wild-type of BY4741 has a certain amount of 2-PE production (1.205g/L). After the introduction of heterogeneous paas gene, the 2-PE production has remarkablely increased. Among them, the production of 2-PE produced by the yeast strain which was introduced petunia-paas increased the most(1.570g/L), followed by the strain which was introduced vanda-paas (1.514g/L) BBa_K3753001 and rosa-paas (1.341g/L) BBa_K3753002.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 60
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 60
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 60
  
• 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

        Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA, consisting of dSpCas9, CDA, and UGI. It was first registered in 2020. In order to test the editing efficiency of this composite part, we construct the dual plasmid system based on the (BBa_K3645011). We selected the cadA, maeA, and maeB genes as the testing sites, and the related pTarget plasmids were constructed. Results showed that the (BBa_K3645011) can successfully work in the BL21 (DE3), and the editing efficiency of single gene editing, double genes editing and triple genes editing can reach 85%, 56% and 25%, respectively (Fig. 1). These results provide references for future iGEM teams to choose gene-editing tools in E.coli.

Fig.1 The gene editing efficiency of the part of dCas9-CDA-UGI.