Difference between revisions of "Part:BBa K3645011"
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===Characterization from iGEM21-NNU-China===
 
===Characterization from iGEM21-NNU-China===
  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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 (<partinfo>BBa_K3645011</partinfo>). We selected the cadA, maeA, and maeB genes as the testing sites, and the related pTarget plasmids were constructed. Results showed that the (<partinfo>BBa_K3645011</partinfo>) 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.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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 (<partinfo>BBa_K4297067</partinfo>) 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.
 
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:'''Fig.1 The gene editing efficiency of the part of dCas9-CDA-UGI.'''  
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:'''Fig. 1. A. The key synthetic steps of the Ehrlich pathway. B. Overexpression of PhPAAS enhances 2-PE yield.'''  
 
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<p><b><h2>Reference</h2></b></p>
 
<p><b><h2>Reference</h2></b></p>
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<p>1. Zhao D, Li J, Li S, Xin X, Hu M, Price MA, Rosser SJ, Bi C, Zhang X. Glycosylase base editors enable C-to-A and C-to-G base changes. Nature Biotechnology. 2021; 39: 35–40.</p>
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<p>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.</p>
  
 
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Revision as of 11:53, 13 October 2022


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 (No part name specified with partinfo tag.) 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.