Part:BBa_K5246037

HMS147(DE3) ΔWecA

Introduction

Vilnius-Lithuania iGEM 2024 project Synhesion aspires to create biodegradable and environmentally friendly adhesives. We were inspired by bacteria, which naturally produce adhesives made from polysaccharides. Two bacteria from aquatic environments - C. crescentus and H. baltica - harness 12 protein synthesis pathways to produce sugars, anchoring them to the surfaces. We aimed to transfer the polysaccharide synthesis pathway to industrially used E. coli bacteria to produce adhesives. Our team concomitantly focused on creating a novel E. coli strain for more efficient production of adhesives.

Strain description

Escherichia coli HMS174(DE3) protein expression strain alternative to BL21(DE3) and is derived from E. coli K-12[1]. It produces recombinant proteins under the control of T7 RNA polymerase. Unlike BL21(DE3), the HMS174(DE3) strain can metabolize galactose [2],[3].

Table 1. Genotype of HMS174(DE3) E. coli Strain

Genotype	F- recA1 hsdR(rK12- mK12+) (DE3) (RifR)

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.HMS174(DE3) strain is an alternative expression strain to BL21(DE3). Retains the lon protease, which can degrade heterologous proteins. More efficient at expressing non-prokaryotic proteins with rare codons due to its different codon usage bias.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 301
  Illegal XbaI site found at 1523
  Illegal PstI site found at 958
  Illegal PstI site found at 1545
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 958
  Illegal PstI site found at 1545
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1171
  Illegal BamHI site found at 1557
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 301
  Illegal XbaI site found at 1523
  Illegal PstI site found at 958
  Illegal PstI site found at 1545
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 301
  Illegal XbaI site found at 1523
  Illegal PstI site found at 958
  Illegal PstI site found at 1545
  Illegal NgoMIV site found at 507
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

genotype

F- recA1 hsdR(rK12- mK12+) (DE3) (Rif R)(KanR) ΔwecA