Team:BHSF ND/Project/Toxin
2019 IGEM BHSF_NDIndex
1. Background
2. Design
3. Discussion
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Wetlab
Leakage
Bistable System
Recombinase
Toxin
Background
Toxin proteins are good materials for detecting leakage of inducible promoters. Because sometimes a molecule of translated toxin, it can have a great impact on cell growth.
Design
To further test the leakage effect, we incorporate toxin protein—Kid, ParE and Colicin—into the plasmid. ParE toxins are DNA gyrase (Gyr) inhibitors.【1】 Toxin Kid which closely resembles the DNA gyrase-inhibitory toxin protein CcdB from E. coli can inhibit DNA replication. 【2】 Toxin Colicin binds to outer membrane receptors, using them to translocate to the cytoplasm or cytoplasmic membrane, where they exert their cytotoxic effect, including depolarization of the cytoplasmic membrane, DNase activity, RNase activity, or inhibition of murein synthesis.
We chose pBAD as the inducible promoter in this experiment for it has lower basal leakage level than XylS. We set all together four different group including one control. We measured OD 600 value which indicate cell growth for the following seven hours.
Experiment & Results
At the first level of experiment we incorporate toxins with pBAD&AraC. At the absence of inducer, all of the three toxin experiment group demonstrated a lower OD value with more significant low OD value in BN062（with toxin ParE）and BN065 (with toxin Colicin) than control. Cell growth was affected in each experimental group. When measured after 12 hours, the trend is more obvious with BN065 and BN062 demonstrate a OD value of 0.1891 and 0.1604 respectively. Whereas control group is 0.7755. The result suggests that even without inducer, pBAD system have noticeable leakage when incorporated with toxins which may affected cell growth in some cases.
Figure 1: Growth Curve for Toxin plasmid with pBAD system in the period of 7 hours.
Figure 2: Bar graph of OD 600 value for Toxin with pBAD system after 12 hours.
We want to test our bi-stable system to see wether it can really prevent leakage. We construct three toxin-bistable-system plasmid.When compared to BN063(Kid&pBAD) during a seven hours period, we can observe a significant improvement in cell growth in BN084 as OD 600 value have is higher. This suggests that our system has a significant role in inhibiting the expression of toxin proteins. Later, when we inoculated the colonies, we found that the toxin protein integrated into the bistable state was significantly inhibited in expression. Cell growth was significantly better than BN063.
Figure 3: Growth curve for BN063(Kid&pBAD) and BN084(Kid&bi-stable system) in the period of 7 hours.
Figure 5: Colony growth for toxin bistable system after 20 hours.
Discussion
After our design and experimental verification, we proved the availability of toxin in the suicide system. Under the careful reading of iGEM security requirements, we ensure the scientificity, usability and efficiency of the selected toxin. After comparison and screening, we selected the most efficient toxin, Kid, as part of our final conditional suicide system.
References
【1】A RelE/ParE superfamily toxin in Vibrio parahaemolyticus has DNA nicking endonuclease activit
Biochemical and Biophysical Research Communications, 15 July 2017, Pages 29-34
【2】Structural and Functional Analysis of the Kid Toxin Protein from E. coli Plasmid R1. Structure, Volume 10, Issue 10, October 2002, Pages 1425-1433