Difference between revisions of "Part:BBa K4880022"
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-This composite part encodes for the antitoxin sepA1 and toxin sepT1 which are controlled by the light inducible promoter PpsbA2 and Fe3+ ion repressed promoter Pisi7942 respectively. In this PemK-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+.
 
-This composite part encodes for the antitoxin sepA1 and toxin sepT1 which are controlled by the light inducible promoter PpsbA2 and Fe3+ ion repressed promoter Pisi7942 respectively. In this PemK-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+.
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<center><html><img src ="https://static.igem.wiki/teams/4880/wiki/parts/bs-system-diagram.png" width = "50%"><br></html></center>
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<center>Figure 1: Diagram of type II TA system</center>
  
 
<!-- Add more about the biology of this part here
 
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Revision as of 23:32, 11 October 2023


PpsbA2-sepA1-PisiA7942-sepT1

-This composite part encodes for the antitoxin sepA1 and toxin sepT1 which are controlled by the light inducible promoter PpsbA2 and Fe3+ ion repressed promoter Pisi7942 respectively. In this PemK-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 XbaI site found at 490
    Illegal PstI site found at 1261
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1261
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 490
    Illegal PstI site found at 1261
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 490
    Illegal PstI site found at 1261
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 502
    Illegal SapI.rc site found at 476


Assembly

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 PisiA79423 from Synechococcus elongatus PCC 7942.


Figure 2: pPMQAK1-PpsbA2-sepA1-Pisi7942-sepT1 plasmid diagram

Parts

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.

sepA1

sepA1 is an antitoxin that is derived from the large plasmid pANL of Synechococcus 7942.

PisiA7942

This Fe3+ repressed promoter derived from Synechococcus elongatus PCC 7942 allows the toxin to be produced only when there is no Fe3+ present.

sepT1

sepT1 is a toxin that is derived from the large plasmid pANL of Synechococcus 7942.

Results

After transforming pPMQAK1-PpsbA2-sepA1-Pisi7942-sepT1 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-sepA1-Pisi7942-sepT1 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-sepA1-Pisi7942-sepT1 colony PCR gel electrophoresis results (Synechocystis sp. PCC 6803)

After successfully transforming pPMQAK1-PpsbA2-sepA1-Pisi7942-sepT1 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-sepA1-Pisi7942-sepT1 (BS3) 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. There is no apparent increase in cell concertation of the Synechocystis sp. PCC 6803 containing the plasmid pPMQAK1-PpsbA2-sepA1-Pisi7942-sepT1 (BS3), which suggest that the biocontainment system worked.