Difference between revisions of "Part:BBa K2433004"
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| − | The | + | The <i>virB1</i> 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. | 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. | + | 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. Cell growth and RFP expression were quantified by measuring density and fluorescence with a microplate reader. Density was measured using OD600 and fluorescence emission at 608nm was measured following excitation at 587nm. Slight difference in fluorescence was observed between un-induced and induced samples (Figure 1) after 6.5 hours. While preliminary data suggests that this part is inducible by acetosyringone, optimization of assay conditions is necessary for confirmation. |
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| − | <p> <b>Figure | + | <p> <b>Figure 1</b>: The average fluorescence in <i>Agrobacterium</i> with the virB1 promoter-RFP construct in pCAMBIA-MCS that are induced with acetosyringone and without acetosyringone measured at 6 hours. |
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| − | <p><b> Figure | + | <p><b> Figure 2</b> : The average OD600 in <i>Agrobacterium</i> with the virB1 promoter-RFP construct in pCAMBIA-MCSthat are induced with acetosyringone and without acetosyringone measured at 6 hours. |
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| − | Future experiments are needed to validate whether this part is inducible by acetosyringone. The literature offers several explanations for the | + | 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 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 | References | ||
Revision as of 02:08, 2 November 2017
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. Cell growth and RFP expression were quantified by measuring density and fluorescence with a microplate reader. Density was measured using OD600 and fluorescence emission at 608nm was measured following excitation at 587nm. Slight difference in fluorescence was observed between un-induced and induced samples (Figure 1) after 6.5 hours. While preliminary data suggests that this part is inducible by acetosyringone, optimization of assay conditions is necessary for confirmation.
Figure 1: The average fluorescence in Agrobacterium with the virB1 promoter-RFP construct in pCAMBIA-MCS that are induced with acetosyringone and without acetosyringone measured at 6 hours.
Figure 2 : The average OD600 in Agrobacterium with the virB1 promoter-RFP construct in pCAMBIA-MCSthat are induced with acetosyringone and without acetosyringone measured at 6 hours.
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 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 118