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| − | The <i>virB1</i> promoter is an inducible promoter for <i>virB1</i> that was taken from the <i>Agrobacterium Tumefaciens</i> plasmid <i>pTiC58</i>. This promoter is induced by acetosyringone and the <i>virB1</i> gene is involved in virulence in wild type <i>A. Tumefaciens</i> (Rogowsky, et al., 1987). Wounded plants release acetosyringone, causing <i>A. Tumefaciens</i> to up-regulate the expression of <i>virB1</i> when near a wound (Brencic, A., & Winans, S. C., 2005). In the context of synthetic biology, the <i>virB1</i> promoter is a useful tool to control gene expression. For example, in <i>A. Tumefaciens</i> the <i>virB1</i> promoter could be used to keep a potentially toxic gene like <i>CRISPR</i> (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 <i>virB</i> induction. <i>VirG</i> and <i>virA</i> are both weakly inducible by acetosyringone (Rogowsky, et al., 1987). | + | The <i>virB1</i> promoter is an inducible promoter for <i>virB1</i> that was taken from the <i>Agrobacterium tumefaciens</i> plasmid <i>pTiC58</i>. This promoter is induced by acetosyringone and the <i>virB1</i> gene is involved in virulence in wild type <i>A. tumefaciens</i> (Rogowsky, et al., 1987). Wounded plants release acetosyringone, causing <i>A. tumefaciens</i> to up-regulate the expression of <i>virB1</i> when near a wound (Brencic, A., & Winans, S. C., 2005). In the context of synthetic biology, the <i>virB1</i> promoter is a useful tool to control gene expression. For example, in <i>A. tumefaciens</i> the <i>virB1</i> promoter could be used to keep a |
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| − | The <i>virB1</i> promoter was cloned into two backbones (<i>K135010</i> and <i>J04650</i>) containing red fluorescent protein (RFP) and transformed into the <i>A. Tumefaciens GV3101</i> for further testing.
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| − | An acetosyringone induction assay was performed using cultured <i>A. Tumefaciens</i> containing the <i>virB1-RFP</i> fusion in the <i>J04650</i> 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 uninduced control. Cell growth and <i>RFP</i> 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.
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| − | [[Image:FluorescenceEvanG.png|450px]][[Image:OD600 EvanG.png|450px]]
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| − | <p> <b>Figure 1: Left Image</b>: The average fluorescence in <i>Agrobacterium</i> with the <i>virB1</i> promoter-<i>RFP</i> construct in <i>pCAMBIA-MCS</i> that was induced with acetosyringone and without acetosyringone measured at 6 hours.
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| − | </p>
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| − | <p><b> Figure 2: Right Image</b> : The average OD600 in <i>Agrobacterium</i> with the <i>virB1</i> promoter-<i>RFP</i> construct in <i>pCAMBIA-MCS</i> that are induced with acetosyringone and without acetosyringone measured at 6 hours.
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| − | </p>
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| − | Future experiments are needed to validate whether this part is 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 <i>virA</i> and <i>virG</i> are only weakly inducible by acetosyringone (Rogowsky, et al., 1987). Further, a previous study suggested that efficient <i>virB</i> induction required <i>virA</i> and <i>virG</i> from the same source plasmid (Krishnamohan, A., Balaji, V., & Veluthambi, K., 2001). The reason being that different plasmids encode different <i>vir</i> boxes (DNA binding regions) inside their promoters (Krishnamohan, A., Balaji, V., & Veluthambi, K., 2001). The <i>virB1</i> promoter part was taken from <i>pTiC58</i>, while <i>virA</i> and <i>virG</i> are derived from the strain <i>GV3101</i>. 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 <i>virB1</i> promoter. It is possible that all of these factors dramatically reduced the induction of the <i>virB1</i> promoter.
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| − | References
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| − | 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.
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| − | 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.
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| − | 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
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| − | 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.
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| − | 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.
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| − | Nollet, L. (2008) Handbook of Meat, Poultry and Seafood Quality. Ames, Iowa: Blackwell Publishing.
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| − | 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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| − | <!-- Add more about the biology of this part here
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| − | ===Usage and Biology===
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| − | <span class='h3bb'>Sequence and Features</span>
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| − | <partinfo>BBa_K2433004 SequenceAndFeatures</partinfo>
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| − | <!-- Uncomment this to enable Functional Parameter display
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| − | ===Functional Parameters===
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| − | <partinfo>BBa_K2433004 parameters</partinfo>
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