Difference between revisions of "Part:BBa K5246036"

 
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WecA is Undecaprenyl-phosphate &#945;-N-acetylglucosaminyl transferase initiating the biosynthesis of enterobacterial common antigen (ECA) and O-antigen PS by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate to form Und-P-P-GlcNAc. WecA is the first glycosyltransferase, and deleting the wecA gene impairs the ECA synthesis pathway in E.coli, therefore lipopolysaccharides containing it are not produced. We wanted to disable the ECA pathway to reduce the metabolic burden on the cell. We aimed to optimize polysaccharide production by creating a more efficient strain. Rosetta(DE3)pLysS strain is designed to overcome the limitations of E. coli's codon usage by providing additional tRNAs for certain codons found in eukaryotic proteins. This allows for better expression of these proteins. In Rosetta(DE3)pLysS, the rare tRNA genes and the T7 lysozyme gene are on the same plasmid.
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===Introduction===
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===Strain description===
  
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===Usage and Biology===
 
===Usage and Biology===
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WecA is Undecaprenyl-phosphate &#945;-N-acetylglucosaminyl transferase initiating the biosynthesis of enterobacterial common antigen (ECA) and O-antigen PS by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate to form Und-P-P-GlcNAc. WecA is the first glycosyltransferase, and deleting the wecA gene impairs the ECA synthesis pathway in E.coli, therefore lipopolysaccharides containing it are not produced. We wanted to disable the ECA pathway to reduce the metabolic burden on the cell. We aimed to optimize polysaccharide production by creating a more efficient strain. Rosetta(DE3)pLysS strain is designed to overcome the limitations of E. coli's codon usage by providing additional tRNAs for certain codons found in eukaryotic proteins. This allows for better expression of these proteins. In Rosetta(DE3)pLysS, the rare tRNA genes and the T7 lysozyme gene are on the same plasmid.
  
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===Sequence and Features===
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<span class='h3bb'>Sequence and Features</span>
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===Functional Parameters===
 
===Functional Parameters===
 
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===Experimental characterization===

Revision as of 14:05, 23 September 2024


Rosetta(DE3)pLysS ΔWecA

Introduction

Strain description

Usage and Biology

WecA is Undecaprenyl-phosphate α-N-acetylglucosaminyl transferase initiating the biosynthesis of enterobacterial common antigen (ECA) and O-antigen PS by catalyzing the transfer of N-acetylglucosamine (GlcNAc)-1-phosphate onto undecaprenyl phosphate to form Und-P-P-GlcNAc. WecA is the first glycosyltransferase, and deleting the wecA gene impairs the ECA synthesis pathway in E.coli, therefore lipopolysaccharides containing it are not produced. We wanted to disable the ECA pathway to reduce the metabolic burden on the cell. We aimed to optimize polysaccharide production by creating a more efficient strain. Rosetta(DE3)pLysS strain is designed to overcome the limitations of E. coli's codon usage by providing additional tRNAs for certain codons found in eukaryotic proteins. This allows for better expression of these proteins. In Rosetta(DE3)pLysS, the rare tRNA genes and the T7 lysozyme gene are on the same plasmid.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 128
    Illegal XbaI site found at 1350
    Illegal PstI site found at 106
    Illegal PstI site found at 693
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 106
    Illegal PstI site found at 693
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 480
    Illegal BamHI site found at 94
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 128
    Illegal XbaI site found at 1350
    Illegal PstI site found at 106
    Illegal PstI site found at 693
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 128
    Illegal XbaI site found at 1350
    Illegal PstI site found at 106
    Illegal PstI site found at 693
    Illegal NgoMIV site found at 1144
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 993
    Illegal SapI.rc site found at 1203

Functional Parameters

genotypeF- ompT hsdSB(rB- mB-) gal dcm (DE3) pLysSRARE (CamR)(KanR) ΔwecA

Experimental characterization