Team:British Columbia/autoinduction
Overview
In the natural life cycle of Agrobacterium, both the conjugation (bacterial cell to bacterial cell plasmid transfer) and virulence (bacterial cell to plant cell plasmid transfer) system are induced by environmental factors. The conjugation system is regulated by a quorum sensing system and responds to molecules secreted by Agrobacterium induced tumours; therefore, the conjugation machinery only happens at certain cell density and Agrobacterium infection. Once the Agrobacterium Ti plasmid is inside a plant cell, it begins the secretion of bacterial metabolites called opines. When these opines diffuse into Agrobacterium cells, traR transcription is initiated. In turn, traR regulates the translation of many other tra proteins (Fuqua et al., 1994). The virulence system is induced off plant produced compounds, to ensure that virulent attack only happens when there is a growing plant nearby (Satchel et. al, 1985). A common Agrobacterium inducer is acetosyringone, a molecule produced by plants when wounded (Ashby et. al, 1987). Acetosyringone interacts with transmembrane vir regions, which in turn induce the transcription of many vir proteins (Winans et. al. 1988). This team sought to employ the natural cell machinery to ensure the potentially damaging CRISPR-Cas9 process would only occur in cases of prevalent Agrobacterium in the environment or pre-existing Agrobacterium infection. For the conjugation or transfer system, we selected two promoters for inquiry: traR and the traA, traC bidirectional promoter (traAC). For the virulence (vir) system, we selected virB1.
Key Achievements
Design
To test the induction of several native Agrobacterium promoters, we synthesized the short sequences upstream of the coding regions flanked with biobricking enzymes, so that for any future endeavours these promoters would be easily accessible to future iGEM teams. We then placed these promoters upstream of RFP coding sequences, then cloned the promoter-reporter fusions into pCAMBIA, the modified Ti plasmid. These constructs were then transformed into Agrobacterium and assayed. The VirB1 promoter was to be assessed for its sensitivity to acetosyringone by measuring RFP production. Likewise, the traR promoter’s activity could be measured in response to induction by opines. Though both promoters are predicted to display activity in response to drug dosage, we expect they will only promote RFP activity inside bacteria with all other functioning transfer or virulence systems, I.e. Agrobacterium which contain the Ti plasmid. Finally, traAC could not be assessed in response to a specific molecule but rather the traR present in the Agrobacterium cells with functioning Ti plasmids producing transfer/conjugation machinery.
Methods
The strains used were Agrobacterium tumefaciens GV3101 and Escherichia coli DH5-Alpha unless otherwise stated. All E. coli cultures were grown in LB media at 37℃ unless otherwise stated. All Agrobacterium were grown in LB at 30℃ unless otherwise stated. All plasmid DNA extractions were performed with ABM Column-Pure Plasmid Mini-Prep Kit. DNA purification from gels with ABM Column-Pure DNA Gel Recovery Kit. The same protocols for DNA gel electrophoresis, restriction enzyme digest, DNA ligation, and chemical and electro transformation were used throughout unless otherwise stated and can be found in the protocols section of the wiki.
Cloning promoters into pSB1C3, BBa_K1357010, and BBa_J04650
We ordered three sequences that we suspected were the promoter sequences from VirB1, TraR, and TraAC from the Ti-plasmid of Agrobacterium tumefaciens and received them in gBlocks from IDT. The promoter fragments, VirB1, TraR, TraAC, and the BioBrick assembly plasmid, pSB1C3, were digested with Eco-RI and Spe-I enzymes and separated via gel purification. We ligated the two fragments together by using T4 ligase. The promoters were also put into BBa_K1357010 and BBa_J04650, reporter plasmids that contain mRFP, with and without a RBS respectively. These reporter plasmids were digested with Xba-I and Pst-I enzymes and the promoters in the pSB1C3 backbone were digested with Spe-I and Pst-I enzymes and ligated.
Transforming plasmids into E.coli S17-1
We transformed the following promoter constructs into chemically competent Escherichia coli S17-1.
We then plated these transformed cells onto LB+CM plates. Two transformants were then picked from each plate to inoculate overnight in the 37 degrees C room. The transformed E.coli were miniprepped and a gel was run to confirm the presence of the correct size bands:
We discovered that the bands did not have the right size and we believe that it was due to the enzyme Pst-I not working correctly. This may have lead to the pCAMBIA plasmid closing on itself and being ligated without being cloned and lead to incorrect band sizes.
Cloning the promoter constructs into pCAMBIA-MCS
Due to time constraints, we picked virB1 and traR to use as promoters and restarted the cloning process. The constructs were digested with Pst-I and Eco-RI. The digests were run on a gel and gel purified in order to isolate the promoter-RFP construct. These constructs were then ligated using T4 ligase into pCAMBIA-MCS that was digested with Pst1 and EcoR1. We then transformed the pCAMBIA-MCS-promoter constructs into electro-competent A. tumefaciens cells using the Electrocompetent Agrobacterium Cell Preparation protocol and plated them on LB+Kan. put picture of gel here
traR did not get colonies after transforming the constructs into pCAMBIA, however there were colonies on the LB+Kan plates of virB1-pCAMBIA construct for both plasmid backbones, BBa_K1357010 and BBa_J04650.
Characterization of virB1 in Agrobacterium
To characterize our promoters and prove that they and were inducible by acetosyringone, performed an assay on the successfully transformed VirB1 constructs. In order to do this, we inoculated three test tubes of each colony in 5mL of LB+Kan of A. tumefaciens colonies that successfully grew on LB+Kan plates overnight. We diluted the overnight A. tumefaciens cultures to an OD of 0.244 and added 200 uL of culture of the different constructs, virB1+J04650 and virB1+K131357010 into a 96 well plate in triplicates. To the triplicates, we then had three treatments - no acetosyringone, 50 uM of acetosyringone, and 100 uM of acetosyringone. We also had a control treatment where only LB+Kan+varying concentrations of acetosyringone (none, 50 uM, 100 uM) were plated on the 96 well plate. We measured the fluorescence every hour up to six hours and then measured one at 30 hours.
We also ran another assay where we took 500 uL of the cultures and inoculated them with 100 uM acetosyringone overnight. We measured the fluorescence of the overnight cultures after 24 hours.
Results
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Conclusion
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References
Fuqua C, Burbea M, Winans SC. Activity of the Agrobacterium Ti plasmid conjugal transfer regulator TraR is inhibited by the product of the traM gene. Journal of Bacteriology. 1995;177(5):1367-1373.
Stachel, S. E., Messens, E., Van Montagu, M., & Zambryski, P. (1985). Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature, 318(6047), 624-629.
Ashby, A. M., Watson, M. D., & Shaw, C. H. (1987). A Ti-plasmid determined function is responsible for chemotaxis of Agrobacterium tumefaciens towards the plant wound product acetosyringone. FEMS microbiology letters, 41(2), 189-192.
Winans, S. C., Kerstetter, R. A., & Nester, E. W. (1988). Transcriptional regulation of the virA and virG genes of Agrobacterium tumefaciens. Journal of Bacteriology, 170(9), 4047–4054.
