Part:BBa_K3823010

MazE antitoxin and Dre recombinase regulated by a hydrogen sulfide switch

Purposes and Design

In our project, we wanted to construct a three-gear kill switch based on hydrogen sulfide sensing, so we needed a component that would allow both MazE and Dre recombinases to be regulated by an H₂S-mediated switch. In this element, MazE and Dre are controlled by Pcstr (BBa_K3823008), which is repressed by CstR(BBa_K3823006).CstR is an HS_nH sensitive regulator. Its position in our plasmid construction is shown here(Figure 1.).

Figure 1. The modified plasmid construction flow chart

Results

We contacted Professor Huaiwei Liu from Shandong University based on literature and fortunately got his help. Using the plasmid he donated as a template, we successfully amplified the CstR-Pcstr sequence(Figure 2A) and used it for our plasmid construction. We successfully constructed the plasmid CstR-Pcstr-MazE -Dre and amplified CstR-Pcstr-MazE-Dre(BBa_K3823010)(Figure 2B).Then we cloned CstR-Pcstr-MazE-Dre to pACYC-rox-ter-rox-MazF.

Figure 2. Agarose electrophoresis

However, according to the sequencing results, we found that Pcstr was missing(not in the PCR product), probably due to the complexity of each block. Meanwhile, we found the terminator between the two rox sites was partly moved after we cultured the final plasmid. We assumed that it could be the leakage of Pcstr that turned on the expression of Dre, which then cut off the terminator. This means we need to optimize our design.But this may prove that our Dre can cut off the sequence between rox sites correctly.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 646
  Illegal PstI site found at 265
  Illegal PstI site found at 953
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 646
  Illegal PstI site found at 265
  Illegal PstI site found at 953
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 646
  Illegal BglII site found at 242
  Illegal BglII site found at 1828
  Illegal BamHI site found at 1006
  Illegal BamHI site found at 2342
  Illegal XhoI site found at 2300
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 646
  Illegal PstI site found at 265
  Illegal PstI site found at 953
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 646
  Illegal PstI site found at 265
  Illegal PstI site found at 953
  
• 1000
  COMPATIBLE WITH RFC[1000]