Difference between revisions of "Part:BBa K2433004"

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VirB1

The VirB1 promoter is an inducible promoter for virB1 that was taken from the Agrobacterium Tumefaciens plasmid pTiC58. This promoter is induced by acetosyringone and the VirB1 gene is involved in virulence in wild type A. Tumefaciens (Rogowsky, et al., 1987). Wounded plants release acetosyringone, causing A. Tumefaciens to up-regulate the expression of VirB1 when near a wound (Brencic, A., & Winans, S. C., 2005). In the context of synthetic biology, the VirB1 promoter is a useful tool to control gene expression. For example, in A. Tumefaciens the virB1 promoter could be used to keep a potentially toxic gene like CRISPR (Peters, J. M., et al., 2015) quiescent until the bacteria neared a plant wound. Functional virG and virA are generally (Powei, B. S. and Kado, C. I., 1990) also required for virB induction. VirG and virA are both weakly inducible by acetosyringone (Rogowsky, et al., 1987)..

The VirB1 promoter was cloned into two backbones (K135010 and J04650) containing red fluorescent protein (RFP) and transformed into the Agrobacterium Tumefaciens strain GV3101 for further testing.

An acetosyringone induction assay was performed using cultured A. Tumefaciens containing the VirB1-RFP fusion in the J04650 backbone. Two overnight cultures were inoculated from the same colony and each culture was split into three tubes for further treatment. Two tubes from each culture were treated with 100uM acetosyringone while the third was used as an un-induced control. Measurements using a microplate reader were taken of the OD600 and ___ fluorescence to quantify cell growth and RFP expression. Samples were measured at 6.5 hours and 23.5 hours, with no significant difference observed between un-induced and induced samples. As such, this part has not yet been experimentally verified as being inducible by acetosyringone.

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Future experiments are needed to validate whether this part is inducible by acetosyringone. The literature offers several explanations for the apparent lack of inducibility in the aforementioned assay. As mentioned above, it has been found that virA and virG are only weakly inducible by acetosyringone (Rogowsky, et al., 1987). Further, a previous study has suggested that for induction of virB to occur efficiently, virA and virG should be from the same source plasmid (Krishnamohan, A., Balaji, V., & Veluthambi, K., 2001). The reason being that different plasmids encode different vir boxes (DNA binding regions) inside their promoters (Krishnamohan, A., Balaji, V., & Veluthambi, K., 2001). The VirB1 promoter part was taken from pTiC58, while virA and virG are derived from the strain GV3101. Lastly, the growth assay listed above was performed using small amounts (<5mL) of culture inside test tubes, while other papers have used larger volumes for similar experiments (Vernade D, Herrera-Estrella A, Wang K, Van Montagu M., 1998). Acetosyringone is listed as a volatile compound (Nollet, L., 2008), making it possible that the increased surface area to volume ratio in our experiment caused the acetosyringone to evaporate before it was able to induce the virB1 promoter. It is possible that all of these factors dramatically reduced, or in the case of volatility, prevented the induction of the VirB1 promoter.

References

Powei, B. S. and Kado, C. I. (1990). Specific binding of VirG to the vir box requires a C- terminal domain and exhibits a minimum concentration threshold. Molecular Microbiology, 4: 2159–2166.

Rogowsky, P. M., Close, T. J., Chimera, J. A., Shaw, J. J., & Kado, C. I. (1987). Regulation of the vir genes of Agrobacterium tumefaciens plasmid pTiC58. Journal of Bacteriology, 169(11), 5101–5112.

Peters, J. M., Silvis, M. R., Zhao, D., Hawkins, J. S., Gross, C. A., & Qi, L. S. (2015). Bacterial CRISPR: Accomplishments and Prospects. Current Opinion in Microbiology, 27, 121–126. http://doi.org/10.1016/j.mib.2015.08.007

Krishnamohan, A., Balaji, V., & Veluthambi, K. (2001). Efficient vir Gene Induction in Agrobacterium tumefaciens Requires virA, virG, and vir Box from the Same Ti Plasmid. Journal of Bacteriology, 183(13), 4079–4089.

Vernade D, Herrera-Estrella A, Wang K, Van Montagu M. (1998). Glycine betaine allows enhanced induction of the Agrobacterium tumefaciens vir genes by acetosyringone at low pH. J Bacteriol. 1988 Dec;170(12):5822-9.

Nollet, L. (2008) Handbook of Meat, Poultry and Seafood Quality. Ames, Iowa: Blackwell Publishing.

Brencic, A., & Winans, S. C. (2005). Detection of and Response to Signals Involved in Host-Microbe Interactions by Plant-Associated Bacteria. Microbiology and Molecular Biology Reviews, 69(1), 155–194. http://doi.org/10.1128/MMBR.69.1.155-194.2005

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