Difference between revisions of "Part:BBa K2433002"

Latest revision as of 03:57, 2 November 2017

ietAS

ietAS is composite part made up of ietA an ietS, the genes encoding the toxin antitoxin system ietAS native to tumor-inducing (Ti) plasmids of Agrobacterium tumefaciens. This part comes from the nopaline type Ti plasmid pTiC58.The ietAS operon was given its name after the incompatibility enhancer of the Ti plasmid. ietAS has been shown in literature to be important in reducing the transconjugant efficiency for different incoming Ti plasmids as well as contributing to the stability of plasmids harboring this construc (Figure 1).

ietAS is a type II toxin/antitoxin system, meaning both ietA and ietS are proteins. In ietAS, the antitoxin ietA is upstream of the toxin ietS. This is typical of type II toxin/antitoxin systems.ietA is less stable, but expressed at higher levels. ietS is more stable, and expressed at lower levels.

ietA neutralizes the effect of ietS on the host cell by forming a protein complex. When a plasmid loses its toxin/antitoxin construct, ietA levels become depleted due to rapid degradation by proteases. This enables ietS to exert its toxicity on the host cell, inhibiting growth. Since cells that lose the plasmid containing the toxin/antitoxin system are terminated, these constructs are useful in selecting for a certain plasmid to be maintained.

ietAS was shown to enhance the incompatibility and stability of the Ti plasmid in Agrobacterium tumefaciens, however, the presence of ietAS showed no effect when tested in E. coli. (Yamamoto et al., 2009)

Figure 1: B) Effect of transconjugation of an incoming plasmid in Agrobacterium with or without theietAS construct. C) Effect on segregational plasmid stability of ietAS construct. A lower CFU ratio indicates a higher plasmid maintenance, demonstrating that ietAS increases plasmid stability. (Yamamoto et al., 2009)

We cloned ietAS into pCambia-MCS plasmid, but were unable to fully characterize its efficacy on the plasmid stability. Due to time restrictions, we were only able to run a plasmid stability assay for 3 days using spot plating to compare the amount of colony forming units on LB versus LB-Kanamycin (Figure 2). Unexpectedly we noticed a decrease in the ratio of CFU's on LB-Kan to CFU's on LB with the ietAS (Figure 3). Hence, we did not see a significant difference between the pCambia plasmid containing ietAS and pCambia plasmid without ietAS. This could be due to the presence of pVS1 StaA on pCambia, a stabilizing protein which is shown to maintain plasmids containing it for 100 generations growing at 30℃ (Heeb et Al., 2000). In future work we could perform the same experiment knocking out the pVS1 staA gene in pCambia. Visit our wiki for more information: http://2017.igem.org/Team:British_Columbia/plasmid_maintenance

Figure 2: Plasmid stability plating assay, day 2. Top-left: pCambia without ietAS in Agrobacterium plated on LB-Kan agar. Bottom-left: pCambia without ietAS in Agrobacterium plated on LB agar. Top-right: pCambia with ietAS in Agrobacterium plated on LB-Kan agar. Bottom-right: pCambia with ietAS in Agrobacterium plated on LB agar. Each sample is plated as two technical replicates over five dilutions based on an OD600 reading.

Figure 3: Effect of ietAS on plasmid stability. CFU ration is the number of CFU's on LB-Kan agar plate divided by the number of CFU's on LB agar plate. See protocols section for details. Each CFU value is the average of three biological replicates, each biological replicate had two technical replicates and was corrected against OD600. Vertical bars represent standard deviation.

References
Heeb, S., Itoh, Y., Nishijyo, T., Schnider, U., Keel, C., Wade, J., Haas, D. (2000). Small, Stable Shuttle Vectors Based on the Minimal pVS1 Replicon for Use in Gram-Negative, Plant-Associated Bacteria. Molecular Plant-Microbe Interactions, 13(2), 232-237. doi:10.1094/mpmi.2000.13.2.232
Melderen, L. V., & Bast, M. S. (2009). Bacterial Toxin–Antitoxin Systems: More Than Selfish Entities? PLoS Genetics, 5(3). doi:10.1371/journal.pgen.1000437
Unterholzner, S. J., Poppenberger, B., & Rozhon, W. (2013). Toxin–antitoxin systems: Biology, identification, and application. Mobile Genetic Elements, 3(5), e26219. http://doi.org/10.4161/mge.26219
Yamamoto, S., Kiyokawa, K., Tanaka, K., Moriguchi, K., & Suzuki, K. (2009). Novel Toxin-Antitoxin System Composed of Serine Protease and AAA-ATPase Homologues Determines the High Level of Stability and Incompatibility of the Tumor-Inducing Plasmid pTiC58. Journal of Bacteriology, 191(14), 4656-4666. doi:10.1128/jb.00124-09

Sequence and Features