Part:BBa_K4880021

PpsbA2-sepA2-PisiA6803-sepT2

This composite part encodes for the antitoxin sepA2 and toxin sepT2 which are controlled by the light inducible promoter PpsbA2 and Fe3+ ion repressed promoter PisiA6803 respectively. In this VapC-like type II TA system, the antitoxin protein neutralizes the toxin via direct protein reaction. The toxin will only be produced without the presence of Fe3+ ion, therefore leading to a programmed suicide in environments lacking Fe3+.

Figure 1: Diagram of type II TA system

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 684
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 684
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 742
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 684
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 684
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 996
  

Plasmid construction

Through homologous recombination, we intergrated this sequence into the broad host range replicative vector pPMQAK1. The antitoxin is expressed using the light inducible promoter PpsbA2 from Synechocystis sp. PCC 6803, and the toxin is expressed using the Fe3+ ion repressed promoter PisiA6803 from Synechocystis sp. PCC 6803.

Figure 2: pPMQAK1-PpsbA2-sepA2-Pisi6803-sepT2 plasmid diagram

PpsbA2

Using a light inducible promoter to control the expression of the antitoxin allows it to be expressed under normal Synechocystis sp. PCC 6803 cultivation conditions.

sepA2

sepA2 is an antitoxin that is derived from the large plasmid pANL of Synechococcus 7942.

PisiA6803

This Fe3+ repressed promoter derived from Synechocystis sp. PCC 6803 allows the toxin to be produced only when there is no Fe3+ present.

sepT2

sepT2 is a toxin that is derived from the large plasmid pANL of Synechococcus 7942.

Results

After transforming pPMQAK1-PpsbA2-sepA2-Pisi6803-sepT2 into E. coli DH5α we performed colony PCR on the monocultures and selected the successfully transformed ones for amplification and extraction to later transform it into Synechocystis sp. PCC 6803. The figure below shows the colony PCR results.

Figure 3: pPMQAK1-PpsbA2-sepA2-Pisi6803-sepT2 colony PCR gel electrophoresis results (E. coli DH5α)

After transforming pPMQAK1-PpsbA2-sepA2-Pisi6803-sepT2 (BS1) into Synechocystis sp. PCC 6803 we performed colony PCR. Below are the results.

Figure 4: pPMQAK1-PpsbA2-sepA2-Pisi6803-sepT2 colony PCR gel electrophoresis results (Synechocystis sp. PCC 6803)

After successfully transforming pPMQAK1-PpsbA2-sepA2-Pisi6803-sepT2 into Synechocystis sp. PCC 6803, we assessed the effectiveness of the suicidal system by growing the engineered cyanobacteria on agar plates with and without Fe3+. We adjusted the samples to OD750=0.1 and further diluted them to 0.01, 0.001 and 0.0001. The diluted samples were dropped onto BG-11 agar plates with Fe3+ ions and BG-11 agar plates without Fe3+ ions. As a control group, engineered Synechocystis sp. PCC 6803 containing our constructed plasmid, αPS, was also diluted and dropped onto both agar plates with and without Fe3+.

Figure 5: Biosafety samples on BG-11 agar plates with and without Fe3+ ion after 1 week of cultivation

Through our first attempt of testing the effectiveness of the biocontainment system in Synechocystis sp. PCC 6803 we were unable to get clear results of whether the cyanobacteria with the biocontainment system died on the agar plate without Fe3+, or the concentration was too low for us to see it. As can be seen on the picture of both agar plates, all Synechocystis sp. PCC 6803 containing pPMQAK1-PpsbA2-sepA2-Pisi6803-sepT2 (BS2) and the control group were able to grow normally on the agar plate with Fe3+ ion and none of the Synechocystis sp. PCC 6803 samples containing the biocontainment system can be seen on the agar plate without Fe3+ ion. However, our control group, αPS, did not grow either. We then realized that cyanobacteria growth is repressed in environments lacking Fe3+, thus the cell concentration must be very low and appears as if it has not grown.

To get more exact results, we tested the biocontainment system in Synechocystis sp. PCC 6803 again by cultivating the cyanobacteria in liquid BG-11 medium with and without Fe3+ ions. Since Synechocystis sp. PCC 6803 grows faster in liquid BG-11 medium, we hoped to see more apparent results from the control group growing in the Fe3+ -deficient medium.

Figure 6: Synechocystis sp. PCC 6803 containing the BS plasmids cultivated in liquid Fe3+ -deficient BG-11 medium

After 5 days of cultivation, we got the above results. We did not get desired results with Synechocystis sp. PCC 6803 containing the plasmid pPMQAK1-PpsbA2-sepA2-Pisi6803-sepT2 (BS2) as there is an obvious increase in cell concentration.