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 is triggered at certain cell densities during Agrobacterium infection of plant cells. Once the Agrobacterium Ti plasmid is inside a plant cell, secretion of bacterial metabolites called opines are activated. At certain concentration of opines inside Agrobacterium cells, traR activator transcription is initiated, which, in turn, activates the translation of other Tra proteins (Fuqua et al., 1994). The virulence system is induced off plant-produced metabolites, to ensure that virulent attack only occurs when there is a growing plant nearby (Satchel et. al, 1985). A common Agrobacterium gene inducer is acetosyringone, a molecule produced by plants when wounded (Ashby et. al, 1987). Acetosyringone interacts with transmembrane virulence (vir) genes, which in turn induce the transcription of many other Vir proteins (Winans et. al. 1988). Our team sought to employ the natural cell induction machinery to specifically activate Cas9 protein expression only in cases of prevalent Agrobacterium in the environment or pre-existing Agrobacterium infection. We selected three promoters - traR, traA/traC bidirectional promoter (traAC) and virB1 to test their ability specifically activate protein expression in our system.
- Successfully cloned virB1-RFP into pCAMBIA-MCS and transformed into Agrobacterium tumefaciens.
- Tested the inductive response of the virB1 promoter in Agrobacterium tumefaciens to acetrosyringone.
- Cloned three different promoter sequences into pSB1C3 and submitted them as BioBrick parts BBa_K2433003, BBa_K2433004, and BBa_K2433005 to the registry.
To test the induction of several native Agrobacterium promoters, such as PtraR, PtraAC, PvirB1 we synthesized the short sequences upstream of the corresponding coding regions flanked with BioBrick Prefix and Suffix so that for any future endeavours these promoters would be easily accessible to future iGEM teams. We placed these promoters upstream of RFP in K1357010 and J04650 parts, then cloned the promoter-reporter fusions into modified pCAMBIA-MCS (more details about plasmid design can be found in the Plasmid Maintenance section of our wiki), a plasmid able to replicate in both E.coli and Agrobacterium. These promoter-reporter constructs were transformed into Agrobacterium and assayed. The virB1 promoter was assessed for its sensitivity to acetosyringone by measuring RFP production. Likewise, the traR promoter’s activity was measured in response to induction by opines. Though both promoters are predicted to display activity in response to inducers dosage, we expect they will only promote RFP activity inside bacteria with all transfer or virulence regulators in place, I.e. Agrobacterium which contain the native Ti plasmid.
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 Column-Pure Plasmid Mini-Prep Kit (ABM). DNA purification from gels with Column-Pure DNA Gel Recovery Kit (ABM). The same protocols for DNA gel electrophoresis, restriction enzyme digest, DNA ligation, and chemical and electrotransformation 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 (544bp), traR (217bp), and traAC (257bp) 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 EcoRI and SpeI enzymes and separated via gel purification. We ligated the two fragments together 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 XbaI and PstI enzymes and the promoters in the pSB1C3 backbone were digested with SpeI and PstI enzymes, gel purified 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, digested with EcoRI and PstI and a gel was run to confirm the presence of the correct size bands:
Virb1 and traR promoters were successfully cloned into the K1357010 and J04650 reporter plasmids.
Cloning the promoter constructs into pCAMBIA-MCS
The PvirB1 + J04650, PvirB1 + K1357010, PtraR + J04650 and PtraR + K1357010 constructs were digested with PstI and EcoRI. 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 which was also was digested with Pst1 and EcoR1. We then transformed the pCAMBIA-MCS-promoter constructs into electrocompetent A. tumefaciens cells using the Electrocompetent Agrobacterium Cell Preparation protocol and plated them on LB+Kan. The constructs were digested and ran on a gel to confirm promoter-RFP insertion .
TraR did not have colonies following transformation of 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.
Figure 1: Digestion of traR-reporter and virB1-reporter constructs in pCAMBIA with EcoR1 (From left to right) 1kb ladder; lane 1: virB1-K1357010 in pCAMBIA; lane 2: virB1-J04650 in pCAMBIA; lane 3: traR-K1357010 in pCAMBIA; lane 4: traR-J04650 in pCAMBIA
Characterization of virB1 in Agrobacterium
To characterize our promoters and prove that they and were inducible by acetosyringone, we 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. Unfortunately, we saw no significant results with this assay.
We redid our assay with the virB1 promoter fused with RFP from J04650. We tested three cultures and tested these constructs by splitting each replicate into three tubes for a total of 9 treatments. Two tubes from each culture were treated with 100 uM acetosyringone while the third was used as an uninduced control.
We were able to clone of traR and virB1 promoters into K1357010 and J04650 backbones upstream of the gene for red fluorescent protein (RFP) successfully (Figure 2). Further subcloning of traR and virB1 promoter-RFP fusion into pCAMBIA was also carried out successfully (Figure 1).
Transformation and subsequent testing of promoter-reporter fusions were carried out for the virB1 promoter fused with RFP from the J04650 construct in A. tumefaciens. Initial subcloning failures in prior steps delayed progress and prevented further construct testing.
The virB1 promoter fused with RFP from J04650 was tested in an acetosyringone-induction assay. 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. Slight difference in fluorescence was observed between un-induced and induced samples (Figure 3). Optimization of assay conditions in necessary to confirm induction of RFP expression by acetosyringone.
Figure 2: Digestion of traR and virB1 in pK1357010 and pJ04650 constructs with EcoR1 and Pst1 (From left to right) 1kb ladder; lane 1: virb1 in K1357010 A; lane 2: virb1 in K1357010 B; lane 3: virb1 in J04650 A; lane 4: virb1 in J04650 B; lane 5: virb1 in J04650 C; lane 6: traR in K1357010 A; lane 7: traR in K1357010 B; lane 8: traR in J04650 A; lane 9: traR in J04650 B. (A,B,C refer to biological replicates)
Figure 3: 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 4 : 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.
Autoinduction is an important tool that can be used to allow inducible control of gene expression. In our project, this could allow for inducible expression of the CRISPR/Cas9 system in high cell densities. When expressed at high levels, CRISPR/Cas9 can have toxic effects on the cell (Peters, J. M., et al., 2015), potentially harming aGROW’s ability to propagate and conjugate its engineered plasmid through the infected soil.
Our team was able to design constructs that fused inducible promoters derived from A. Tumefaciens with the gene for the reporter molecule RFP. We were able to begin to characterize the virB1 promoter’s fusion with RFP J04650, showing preliminary evidence for acetosyringone inducibility as suggested by the literature (Rogowsky, et al., 1987). The weak induction observed in experimentation could be explained as a result of biological mechanism or technical procedure. VirA and virG are generally (Powei, B. S. and Kado, C. I., 1990) both required for virB induction, and each is only weakly inducible by acetosyringone (Rogowsky, et al., 1987). Low levels of these proteins could hamper RFP expression. Further, a previous study has suggested that for induction of virB to occur efficiently, virA and virG should be from the plasmid of origin (Krishnamohan, A., Balaji, V., & Veluthambi, K., 2001). Different plasmids encode different vir boxes (protein-binding regions) inside their promoters (Krishnamohan, A., Balaji, V., & Veluthambi, K., 2001). Our virB1 promoter 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. Our results indicate that the virB1 promoter is inducible by acetosyringone, a plant wound hormone. However, further inquiry and characterization are required to use the virB1 part to quiesce parts such as CRISPR/Cas9.
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