Difference between revisions of "Part:BBa K2433004"

Latest revision as of 03:39, 2 November 2017

VirB1

The virB1 promoter is an inducible promoter for virB1 originated from the Agrobacterium tumefaciens plasmid pTiC58. This promoter is induced by plant metabolite acetosyringone and the virB1 gene essential for DNA transfer from wild type A. tumefaciens into a plant cell(Rogowsky, et al., 1987). Wounded plants release acetosyringone, causing A. tumefaciens to up-regulate the expression of virB1 gene (Brencic, A., & Winans, S. C., 2005). In the context of synthetic biology, the virB1 promoter is a useful tool to control gene expression in A. tumefaciens strains. 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) silent until the bacteria neared a plant wound. Functional VirG and VirA proteins 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 reporter plasmids (K135010 and J04650) containing red fluorescent protein (RFP) and transformed into the A. tumefaciens GV3101 for further testing.

An acetosyringone induction assay was performed using A. tumefaciens culture 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 100µM acetosyringone while the third was used as an uninduced control. Cell growth and RFP expression were quantified by measuring density and fluorescence with a microplate reader. Density was measured using OD600 absorbance and fluorescence emission at 608nm was measured following excitation at 587nm. Slight difference in fluorescence was observed between uninduced and induced samples (Figure 1) after 6 hours. While preliminary data suggests that this part is inducible by acetosyringone, optimization of assay conditions is necessary for confirmation.

Figure 1: Left Image: The average fluorescence in Agrobacterium with the virB1 promoter-RFP construct in pCAMBIA-MCS that was induced with acetosyringone and without acetosyringone measured at 6 hours.

Figure 2: Right Image : The average OD600 in Agrobacterium with the virB1 promoter-RFP construct in pCAMBIA-MCS that are induced and non-induced with acetosyringone measured after 6 hours of acetosyringone addition .

Future experiments are needed to validate whether this part is indeed inducible by acetosyringone. The literature offers several explanations for the weak level of induction in the assay mentioned above. As previously listed, it has been found that virA and virG are only weakly inducible by acetosyringone (Rogowsky, et al., 1987). Further, a previous study suggested that efficient virB induction required virA and virG 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 efficiently induce the virB1 promoter. It is possible that all of these factors dramatically reduced 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

Sequence and Features