Difference between revisions of "Team:British Columbia/conjugation"
Ievdokymenko (Talk | contribs) (References) |
Ievdokymenko (Talk | contribs) (Conclusions) |
||
| Line 134: | Line 134: | ||
<h1 class="section-heading">Conclusion</h1> | <h1 class="section-heading">Conclusion</h1> | ||
<p> | <p> | ||
| − | + | With these results, we were able to begin characterizing RP1 as a vehicle for mobilizing our Ti plasmid-targeting CRISPR system in a population of <i>A. tumefaciens</i>. We demonstrated that Ti plasmid-containing strains of <i>A. tumefaciens</i> can receive RP1 via conjugation from <i>E.coli</i>, express its kanamycin selection marker, and maintain this plasmid. These features support its use as a mobilizing vehicle for the Ti plasmid-targeting CRISPR system. | |
| − | + | We were not able to show RP1 moving from <i>A. tumefaciens</i> into other cells, however. If the conjugation had occurred with high frequency in our assay, we should have observed some kanamycin-streptomycin transconjugants after accounting for the spontaneous streptomycin-resistant mutants in the donor culture. This suggested to us that either <i>A. tumefaciens</i> was incapable of donating RP1, contrary to the literature (Quandt et al. 2014), or that our conjugation reaction conditions were not appropriate. | |
| − | + | <i>A. tumefaciens</i> capacity to receive RP1 readily, express genes on the plasmid, and maintain it in culture indicate that it is a suitable RP1, as indicated in the literature. Instead, we speculate that our conjugation incubation of 4 hours may have been insufficient to allow a significant number of conjugation events. We chose this duration because our mating with an <i>E. coli</i> donor gave confluent transconjugant growth after a 6 hour incubation. In hindsight, <i>E. coli</i> much faster growth rate likely allows for the quicker expression of RP1’s conjugation machinery and more efficient transfer from this host. Now, having isolated a streptomycin-sensitive <i>A. tumefaciens</i> RP1 host, future conjugation assays should be conducted with longer incubations. | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</p> | </p> | ||
</div> | </div> | ||
Revision as of 02:27, 2 November 2017
Overview
To be an effective countermeasure against crown gall disease, our CRISPR system targeting the Ti-plasmid must be capable of spreading efficiently in Agrobacterium tumefaciens bacterial population. We investigated if conjugation, a natural bacterial process, could be potentially used to mobilize our plasmid carrying CRISPR/Cas9 between Agrobacterium cells.
Bacterial conjugation is the process by which a plasmid is spread from a donor cell directly into a recipient cell (Llosa et al. 2002). This DNA transfer occurs through a pilus-like structure on the exterior surface of the donor cell. This pilus inserts itself into the recipient when the two cells come into contact, forming a direct conduit. Conjugative transfer requires the donor cell to encode and express the conjugative machinery, including the pilus. Often, this machinery is carried on the plasmid it interacts with, allowing the plasmid to spread itself through a population of cells. The Ti-plasmid itself uses conjugation to spread through the A. tumefaciens population in soil environments and inside crown gall tumour tissue. We considered exploiting this conjugative machinery for delivering our CRISPR system, but this idea was abandoned because the Ti-plasmid’s conjugation genes are strictly regulated. This regulation includes sensory systems that recognize small molecules produced by wounded plants and quorum-sensing systems that respond to high densities of Ti-plasmid in the cell population (Lang and Faure 2014, Subramoni et al. 2014, White and Winans 2007). Consequently, the Ti-plasmid’s conjugative machinery is adapted to conditions where plants are extremely susceptible to infection, conditions that are not ideal for an intervention designed to destroy Ti-plasmids before they can begin the disease process.
We identified an alternate conjugative plasmid called RP1 with several merits for our particular application. It belongs to a broad host range family of plasmids recognized for their capacity to readily propagate antibiotic resistance genes through diverse communities of bacteria. Their conjugation machinery is encoded on the plasmid, allowing it to mobilize itself from a variety of hosts, and this machinery is also not under stringent conditional regulation (Zatyka and Thomas 1998, ). Crucially, we found examples in the literature of RP1 and related plasmids conjugating between and within species of Agrobacterium (Quandt et al. 2004), making it an attractive candidate to test as a vehicle for mobilizing our Ti-plasmid-targeting CRISPR system.
We performed conjugative mating assays to characterize RP1’s capacity for conjugative transfer into Ti plasmid-containing A. tumefaciens recipient cells. We also characterized RP1’s ability to conjugate from A. tumefaciens donors to other A. tumefaciens recipients. We demonstrated that Ti plasmid-containing A. tumefaciens can readily receive RP1 by conjugation from E. coli donors, but failed to observe RP1 conjugation into an A. tumefaciens recipient from an A. tumefaciens donor. Future experiments will demonstrate if A. tumefaciens donors require longer conjugation incubations to fully express conjugation machinery due to their slower growth.
Key Achievements
Methods
RP1 conjugation from E. coli to A. tumefaciens
To investigate the ability of Agrobacterium to act as a RP1 plasmid recipient, we transformed RP1 into E. coli host and performed a conjugative mating reaction with Agrobacterium tumefaciens strain GV3101. In this assay, a donor culture of E. coli containing RP1 was grown in LB supplemented with kanamycin (75 ug/ml), and a recipient culture of A. tumefaciens strain GV3101 was grown in LB supplemented with gentamicin (100 ug/ml). The cultures were harvested at OD 0.250, mixed together at various ratios, concentrated by centrifugation to facilitate cell-to-cell contact, and incubated to allow for conjugative transfer of RP1. Transconjugants were selectively grown by plating the mating reactions on LB agar plates supplemented with both kanamycin (75 ug/ml) and gentamicin (75 ug/ml). This allowed only for growth of A. tumefaciens cells where the kanamycin selection marker on RP1 had moved into recipient A. tumefaciens cells, which carry a gentamycin marker. To control for the possibility of false-positives from spontaneous kanamycin and gentamicin mutations, donor and recipient cultures were separately concentrated by centrifugation, incubated, and plated on selective medium to ensure the absence of growth.
RP1 conjugation between A. tumefaciens
To investigate the ability of RP1 to conjugate between Agrobacterium cells, we designed another conjugation similar to the one above. We grew a donor culture of A. tumefaciens strain GV3101 carrying RP1 plasmid (obtained from the mating assay above) in LB supplemented with kanamycin (75 ug/ml), and a recipient culture of A. tumefaciensstrain LBA4404 in LB supplemented with streptomycin (2000 ug/ml). Cultures were harvested at OD 0.250 and concentrated to OD 2.50 by centrifugation, mixed together in equal proportion, and incubated for 24 hours. Following the incubation, a ten-fold dilution series was prepared from the mating reaction, and aliquots of each dilution were plated on LB agar supplemented with kanamycin (75 ug/ml) and streptomycin (2000 ug/ml) for selective growth and CFU enumeration of transconjugants. In parallel, aliquots of the mating reaction dilutions were plated on LB agar supplemented with gentamicin (100 ug/ml) and streptomycin (2000 ug/ml) for selection and CFU enumeration of transconjugants arising from background Ti plasmid transfer. Control reactions of donor and recipient cultures were plated on the same conditions to ensure the absence of spontaneous kanamycin and streptomycin resistant mutants.
Results
Discussion of conjugation assay of E.coli and GV3101:
The plates with both selection markers (kanamycin (kan) selects for RP1 plasmid and gentamycin (gent) selects for the Agrobacterium recipient) showed growth from all mating reaction of different ratios of donor (E. coli) and recipient GV3101 cells. Our positive controls plated on LB showed growth for both donor and recipient cultures. The negative controls with LB + kan/gent showed no signs of growth for either E.coli and GV3101 strains indicating that E.coli(donor) and GV3101 (recipient) did not develop any spontaneous mutations for kan and gent resistance respectively. Taken together, this indicates that Agrobacterium colonies on the double-selection plates from the mating reaction are successful RP1 transconjugants, and not spontaneous kanamycin or gentamicin resistant mutants from the donor or recipient cultures. Growth on the double-selection marker plates indicated that RP1 plasmid got successfully transferred from E.coli into Agrobacterium GV3101 cells during the mating reactions.
The different donor and recipient concentration ratios in the matings all showed a similar amount of growth on the kan/gent plates, [suggesting mating concentration do not matter in the context of conjugation??].
Discussion of Conjugation Assay of GV3101 + RP1 and LBA4404
The plates with both selection markers for RP1 conjugation, where kan selects for RP1 plasmid and strep selects for recipient, showed growth from all mating reaction up to a dilution of 10^-2. The plates with both selection markers for Ti conjugation, where kan selects for RP1 plasmid and gent selects for recipient, showed growth from the mating reaction with no dilution. Our positive controls plated on LB showed growth for both donor and recipient cultures. The negative controls for recipient on a plate with LB + kan/strep and a plate with LB + gent/strep showed no signs of growth. However, the negative control for donor on the same plate combinations as the recipient showed signs of growth, albeit less growth than the donor positive control even though it was plated with a smaller donor concentration.
Given that the negative controls for our donor (GV3101 + RP1) showed growth, we cannot conclusively suggest anything about RP1 conjugation. Through further investigation, we hypothesized that the streptomycin concentration was too low. [but that wasn’t the case, b/c with higher concentration of strep (2000ug/L) they were still growing]
[Actually we were dealing GV3101 culture with mixture of strep resistant mutants][Explain how we dealt with this]
Complications of Using an Antibiotic Selection Marker on a Hypothetically Mobile Plasmid
In the conjugation assay to test if A. tumefaciens could act as a donor, our recipient (LBA4404) is similarly resistant to all antibiotics with our donor (GV3101), except for its streptomycin marker. However, this marker is located on the Ti plasmid, (which should only be mobile when agro is exposed to ?opines?) and so could hypothetically conjugate from recipient to donor. This would create transconjugants that would be streptomycin and gentamycin resistant, which is the same combination of antibiotic resistance we would expect to see for RP1 conjugation that we actually want to assay.
We hypothesized that the amount of Ti conjugation would be negligible or at least little enough (< 10% of RP1 transconjugants) so that we could measure the RP1 conjugation, and then discount that number by the amount of Ti transconjugants observed. To measure the amount of Ti conjugation from recipient to donor, we also plated the mating reaction onto plates with strep/gent (strep resistance coming from conjugated Ti plasmid and gent resistance inherent in donor genome). There was a possibility that some of the donor and recipients developed spontaneous gent/strep mutations. So, we plated donor and recipient cultures onto gent/strep separately to observe a lack of growth.
Conclusion
With these results, we were able to begin characterizing RP1 as a vehicle for mobilizing our Ti plasmid-targeting CRISPR system in a population of A. tumefaciens. We demonstrated that Ti plasmid-containing strains of A. tumefaciens can receive RP1 via conjugation from E.coli, express its kanamycin selection marker, and maintain this plasmid. These features support its use as a mobilizing vehicle for the Ti plasmid-targeting CRISPR system. We were not able to show RP1 moving from A. tumefaciens into other cells, however. If the conjugation had occurred with high frequency in our assay, we should have observed some kanamycin-streptomycin transconjugants after accounting for the spontaneous streptomycin-resistant mutants in the donor culture. This suggested to us that either A. tumefaciens was incapable of donating RP1, contrary to the literature (Quandt et al. 2014), or that our conjugation reaction conditions were not appropriate. A. tumefaciens capacity to receive RP1 readily, express genes on the plasmid, and maintain it in culture indicate that it is a suitable RP1, as indicated in the literature. Instead, we speculate that our conjugation incubation of 4 hours may have been insufficient to allow a significant number of conjugation events. We chose this duration because our mating with an E. coli donor gave confluent transconjugant growth after a 6 hour incubation. In hindsight, E. coli much faster growth rate likely allows for the quicker expression of RP1’s conjugation machinery and more efficient transfer from this host. Now, having isolated a streptomycin-sensitive A. tumefaciens RP1 host, future conjugation assays should be conducted with longer incubations.
References
Adamczyk M, Jagura-Burdzy G. Spread and survival of promiscuous IncP-1 plasmids. Acta Biochim Pol. 2003;50(2):425-53.
Cook DM, Farrand SK. The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders. J Bacteriol. 1992 Oct;174(19):6238-46.
Lang J, Faure D. Functions and regulation of quorum-sensing in Agrobacterium tumefaciens. Front Plant Sci. 2014 Jan 31;5:14. doi: 10.3389/fpls.2014.00014. eCollection 2014.
Llosa M, Gomis-Rüth FX, Coll M, de la Cruz Fd F. Bacterial conjugation: a two-step mechanism for DNA transport. Mol Microbiol. 2002 Jul;45(1):1-8.
Quandt J, Clark RG, Venter AP, Clark SR, Twelker S, Hynes MF. Modified RP4 and Tn5-Mob derivatives for facilitated manipulation of large plasmids in Gram-negative bacteria. Plasmid. 2004 Jul;52(1):1-12.
Subramoni S, Nathoo N, Klimov E, Yuan ZC. Agrobacterium tumefaciens responses to plant-derived signaling molecules. Front Plant Sci. 2014 Jul 8;5:322. doi: 10.3389/fpls.2014.00322. eCollection 2014.
White CE, Winans SC. Cell-cell communication in the plant pathogen Agrobacterium tumefaciens. Philos Trans R Soc Lond B Biol Sci. 2007 Jul 29;362(1483):1135-48.
Zatyka M, Thomas C. Control of genes for conjugative transfer of plasmids and other mobile elements. FEMS Microbiol Lett, Feb 1998;21(4): 291–319. Doi: 10.1111/j.1574-6976.1998.tb00355
