Part:BBa_K3875027

1.Design

We designed the part(BBa_K3875027) to characterize mazE and mazF. We performed experiment to work our final genetic circuits and characterized mazE and mazF.
This experiment consists of the four parts below:
1.Obtain the mazEF gene and the vgb gene
2.Build a gene expression vector
3.Import the gene expression vector to detect the properties of engineered bacteria

2.Methods

1.Design primers with a homologous arm, using pcr to obtain the target genes mazEF and vgb from E. coli BW25113
2.The expression vector pZE is constructed by the method of enzymatic enzyme connection
3.Validate build results and sequencing
4.Import the constructed expression vector into nissle 1917 observation

3.Results

After the culture and sequencing verification of pZE-pvgb, the constructed plasmids will be imported into nissle 1917, tighten the lid to ensure low oxygen status, 37 degrees Celsius shaker culture 12-16h, found that the colonies grow and the size is obvious, after the extraction of plasmids sent sequencing found lac promoter loss, indicating that the construction of suicide system is not successful

Figure. The growth of bacteria imported into the suicide system

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 450
  Illegal EcoRI site found at 665
  Illegal XbaI site found at 87
  Illegal XbaI site found at 941
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 450
  Illegal EcoRI site found at 665
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 450
  Illegal EcoRI site found at 665
  Illegal BamHI site found at 738
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 450
  Illegal EcoRI site found at 665
  Illegal XbaI site found at 87
  Illegal XbaI site found at 941
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 450
  Illegal EcoRI site found at 665
  Illegal XbaI site found at 87
  Illegal XbaI site found at 941
  
• 1000
  COMPATIBLE WITH RFC[1000]