Part:BBa_K5226052

321(C+cysNC) backbone

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BamHI site found at 2528
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal NgoMIV site found at 2981
  Illegal NgoMIV site found at 3866
  Illegal NgoMIV site found at 4977
  Illegal NgoMIV site found at 5101
  Illegal AgeI site found at 2037
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal SapI site found at 2103
  Illegal SapI.rc site found at 2171
  

Introduction

The existing methods for large-scale production of P34HB primarily rely on microbial fermentation. A key limiting factor in this process is the molar ratio of 4HB. Increasing the 4HB molar ratio can lead to a decrease in the melting temperature and apparent fusion heat of the copolymer, as well as an improvement in the polymer's deformation resistance. Therefore, enhancing the molar ratio of 4HB is crucial for the modification of P34HB.

Usage and Biology

To stabilize cell dry weight while increasing the 4HB molar ratio, we investigated the effect of the pSEVA321 on cell dry weight. We hypothesized that the addition of chloramphenicol during the fermentation process impacted cell growth. Consequently, we decided to adjust the screening pressure and knock out cysNC, a gene encoding a key enzyme in the sulfate assimilation pathway, in Halomonas TD to block the supply of sulfur sources for methionine synthesis. Simultaneously, we incorporated the cysNC gene into the pSEVA321 backbone to screen for strains that had been successfully transformed with the 321-porin194-4hbd-sucD-ogdA-porin194-orfZ plasmid.
We used CRISPR/Cas9 for gene knockout. Due to the challenges of gene editing in Halomonas TD, we employed two methods to block the expression of cysNC. The first method involved knocking out all codons from the start codon (ATG) to the stop codon. The second approach entailed knocking out the core region from its full-length sequence. We designed two sets of gRNAs targeting the specific regions for each knockout method. Both methods were performed simultaneously, and successful knockouts were selected for the subsequent fermentation step.
This part is the pSEVA321 backbone with the cysNC gene added.