Part:BBa_K3875026

1.Design：

We designed the part(BBa_K3875026) 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 phyb gene
2.Build a gene expression vector
3.Import the gene expression vector to detect the properties of engineered bacteria

2.Methods:

Design primers with a homologous arm, using pcr to obtain the target genes mazEF and phyb from E. coli BW25113, the pcr program is shown in the figure
Figure1.Pcr program




Figure. Obtain mazE and mazF gene



2.Connecting five gene fragments using gibson assembly method

Figure. Gibson program



3.Verify the length of the expression vector

Figure. Verify the length of the expression vector




Figure. Enzyme cut validation results



4.The expression carrier is imported into the Nissle 1917 receptor state and cultured under oxygen-free conditions to culture 48h

Figure.The engineered bacteria are cultured under oxygen-free conditions



5.Observe the results and verify them

3.Results

Cultured 16h under aerobic conditions, no colonies were found to grow, which proved that there was no escape of engineering bacteria under aerobic conditions, which is in good compliance with the safety principle.

Figure．Aerobic conditions cultured 16h, engineering bacteria died



After constantly groping for reaction conditions, it was found that Nissle 1917 grew very slowly in an oxygen-free environment, requiring 48-72h growth in order to see the obvious colony of the crucible in the aerobic air environment, it is not possible to observe obvious traces of engineering bacteria growth, which can indicate the success of the suicide system.

Figure. The culture contrast between aerobic environment and anaerobic environment

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 87
  Illegal XbaI site found at 967
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 764
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 87
  Illegal XbaI site found at 967
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 87
  Illegal XbaI site found at 967
  
• 1000
  COMPATIBLE WITH RFC[1000]