Part:BBa_K4119008

Plac-ΔperR

The upper and lower homology arms (HR-UP, HR-DOWN) were constructed at about 500 bp upstream and downstream of the PerR sequence of the Clostridium tyrobutyricum genome, plus a lactose-inducing promoter and a spacer sequence derived from the PerR , generating the CRISPR array .Then inserted it into the pMTL82151 plasmid, the successful plasmid is transformed into E. coli CA434, and introduced into Clostridium tyrobutyricum, by lactose induction to make the spacer hit the perR sequence on the target genome, thereby utilizing the endogenous CRISPR-Cas system knocks the perR out , eliminates the impediment effect of perR on the growth of Clostridium butyrate, thereby achieving aerobic growth of Clostridium tyrobutyricum.

Short Description: lactose-inducing promoter+ CRISPR array

The upper and lower homology arms (HR-UP, HR-DOWN) were constructed at about 500 bp upstream and downstream of the PerR sequence of the Clostridium tyrobutyricum genome, plus a lactose-inducing promoter and a spacer sequence derived from the PerR , generating the CRISPR array .Then inserted it into the pMTL82151 plasmid, the successful plasmid is transformed into E. coli CA434, and introduced into Clostridium tyrobutyricum, by lactose induction to make the spacer hit the perR sequence on the target genome, thereby utilizing the endogenous CRISPR-Cas system knocks the perR out , eliminates the impediment effect of perR on the growth of Clostridium butyrate, thereby achieving aerobic growth of Clostridium tyrobutyricum.

Result

As can be seen from the sequencing results, the perR fragment has been knocked out.

Fig2- Sequencing verification of obtained mutant. The red part represents the absence of the sequence.

At different rotational speeds, the growth of the plasmid knockout strain was better than that of the control strain

Fig3- The growth profiles of Ct (ΔperR) and Ct (Control) under different contents of oxygen.

Fig4- Physiological characteristics of Ct (ΔperR) and Ct (Control) in the presence of oxygen. (A) The intracellular ROS levels in Ct (ΔperR) and Ct (Control) in the presence of oxygen. ROS levels were measured in the logarithmic prophase. (B) The ratio of NADH/ NAD+ in Ct (ΔperR) and Ct (Control) in the presence of oxygen. Phase I, II and III refer to the early-, mid- and late- logarithmic phase, respectively.

Fig5- The survival rate of Ct (ΔperR) and Ct (Control) to oxidative stress.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 368
  Illegal PstI site found at 1597
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 368
  Illegal PstI site found at 1597
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 368
  Illegal BglII site found at 428
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 368
  Illegal PstI site found at 1597
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 368
  Illegal PstI site found at 1597
  
• 1000
  COMPATIBLE WITH RFC[1000]