Part:BBa_K3823012

hydrogen sulfide sensitive, three-gear adjustable kill switch

Design

Biosafety has always been an important part in iGEM. To prevent our engineered bacteria from leaking into the environment, we designed a H₂S sensitive three-gear kill switch to ensure this. H₂S is an important factor in the working environment of our engineered bacteria, so we chose to use H₂S concentration to control the on-off of the kill switch. By searching for the biosensors of various gas molecules, we found a HS_nH-sensing biosensor (the product of SQR oxidized H₂S happens to be HS_nH) -- a gene regulator, CstR, which can sense HS_nH and turn on the expression of the downstream genes (Figure 1.)^[1].

Figure 1. the schematic presentation of CstR. PR(Pcstr): the promoter which CstR can combine with and inhibit; PL: the constitutive promoter which regulate the expression of CstR

If we design a two-gear switch, namely, the switch is off when H₂S concentration is high and on when H₂S concentration is low, then we need to maintain high concentration of H₂S in the culture environment, but this is obviously not realistic. Therefore, we designed a three-gear kill switch based on the Dre/rox recombinant enzyme system ^[2](referring to the work of the [http://2017.igem.org/Team:Edinburgh_UG Edinburgh UG in 2017] and the MazEF toxin-antitoxin system^[3], which can achieve more flexible regulation of the survival of our engineered bacteria. (Figure 2.)

Figure 2. The design of our three-gear kill switch

Under the culture condition, H₂S concentration is not high enough to turn on any of the three pathways, so the bacteria will grow normally. When the concentration of H₂S is high, H₂S will remove the inhibition of CstR on the downstream gene expression by combining with it, then Dre will be expressed, and the terminator before MazF will be removed, resulting in the expression of MazF toxin. Meanwhile, due to the expression of the antitoxin MazE, the bacteria will not die. When working bacteria leak into the environment, H₂S concentration in the environment will return to the normal (low) level, the inhibition of CstR will not be removed, and MazE will no longer be expressed. At this time, because MazF has been expressed, a large amount of MazF will kill the bacteria over time. (Figure 3.)

Figure 3. The work circuit of our kill switch

Experiments

Plasmid construction

Considering the accessibility of the template and the time limitation, we adopted the strategy of assembling the functional components separately obtained by gene synthesis. Considering plasmid incompatibility, we chose pACYC as the vector for this part, which has a different replication system from pET-3a. Plasmids were constructed by restriction enzyme digestion and ligation and In-Fusion. The construction process we designed is shown below.

Figure 4. The initial plasmid construction flow chart

Unfortunately, the synthesis of J23119-CstR-Pcstr-MazE -Dre encountered difficulty so that we couldn't obtain the sequence of J23119-CstR-Pcstr. Considering this part was very necessary for our work, 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 6A) and used it for our plasmid construction.

Figure 5. The modified plasmid construction flow chart

We successfully constructed the plasmid CstR-Pcstr-MazE -Dre and amplified CstR-Pcstr-MazE-Dre(Figure 6B).Then we cloned CstR-Pcstr-MazE-Dre to pACYC-rox-ter-rox-MazF.

Figure 6. Agarose electrophoresis

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1300
  Illegal PstI site found at 919
  Illegal PstI site found at 1607
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1300
  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal PstI site found at 919
  Illegal PstI site found at 1607
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1300
  Illegal BglII site found at 896
  Illegal BglII site found at 2482
  Illegal BamHI site found at 36
  Illegal BamHI site found at 543
  Illegal BamHI site found at 1660
  Illegal BamHI site found at 2996
  Illegal XhoI site found at 2954
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1300
  Illegal PstI site found at 919
  Illegal PstI site found at 1607
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1300
  Illegal PstI site found at 919
  Illegal PstI site found at 1607
  
• 1000
  COMPATIBLE WITH RFC[1000]