Part:BBa_K5291037

pAB-hok/sok

We construct the hok/sok system to realize plasmid anti-loss as well as bacteria suicide when they are out of the mangroves.

Usage and Biology

We use this system to prevent plasmid loss and conduct bacteria suicide.


Fig.1 The schema of the hok/sok system.

And we successfully construct the strain with plasmid pAB-hok/sok.


Fig.2 The AGE result of pAB1-hok/sok.

To verify the function of plasmid anti-loss, we take advantage of the strains with and without hok/sok system, cultivating them in non-resistent LB broth to deliberately induce plasmid loss. After that we transfer them to the non-resistent plates and then select single colonies, scribing them on resistance plates to examine if they still own the plasmid we introduce. We find that after several generation passing the death rate of the groups without hok/sok system is significantly higher than the one with the system, which means that the system is beneficial to plasmid maintaining.


Fig.3 The 17th LB-Amp plate of the verification of plasmid anti-loss function. (Left: hok/sok+ Right: hok/sok-)


Fig.4 The diagram shown the death rate of bacteria in two groups.

We verify the plasmid anti-loss function of hok/sok system.

We also conduct the experiment to examine the function of cell suicide when there is no activator of P_opdH. Since citrate, the main activator of P_opdH, will be taken up by Pseudomonas aeruginosa as the carbon source, we add another promoter pLac which can be activated by IPTG, a kind of substance stable in cells, in front of sok just for experimental research. We devide the bacteria with pAB-hok/sok into two groups, cultivating in the LB-Amp broth with and without IPTG, and testing OD600 of each group after cultivation.


Fig.5 The contrast between the groups with and without IPTG and the diagram of the different OD600 of the two groups.

We can know from the diagram that the OD600 of the group with IPTG is much higher than the other group, which means the better growth of the former group. We successfully verify the specific control of the hok/sok system.
*The concentration of IPTG we choose is 0.6mM, which is found as the best concentration of induction in our pre-laboratory.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 146
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 16
  
• 1000
  COMPATIBLE WITH RFC[1000]