Difference between revisions of "Team:British Columbia/conjugation"
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<h1 class="section-heading">Conjugation Background</h1> | <h1 class="section-heading">Conjugation Background</h1> | ||
<p> | <p> | ||
| − | To be an effective countermeasure against crown gall disease, our CRISPR system targeting the Ti-plasmid must be capable of | + | To be an effective countermeasure against crown gall disease, our CRISPR system targeting the Ti-plasmid must be capable of spreading efficiently in <i>Agrobacterium tumefaciens</i>population. We investigated a natural bacterial conjugation process for this purpose. |
Bacterial conjugation is the process by which a plasmid is spread from a donor cell directly into a recipient cell (). 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. | Bacterial conjugation is the process by which a plasmid is spread from a donor cell directly into a recipient cell (). 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 | + | The Ti-plasmid itself uses conjugation to spread through the <i>A. tumefaciens</i> 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 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 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 <i>Agrobacterium</i> (Quandt et al. 2004), making it an attractive candidate to test as a vehicle for mobilizing our Ti-plasmid-targeting CRISPR system. |
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<h1 class="section-heading">Methods</h1> | <h1 class="section-heading">Methods</h1> | ||
<p> | <p> | ||
| − | RP1 conjugation from E. coli to A. tumefaciens | + | RP1 conjugation from <i>E. coli</i> to <i>A. tumefaciens</i> |
| − | To investigate the ability of Agrobacterium to act as a RP1 recipient, we | + | To investigate the ability of <i>Agrobacterium</i> to act as a RP1 plasmid recipient, we transformed RP1 into <i>E. coli</i> host and performed a conjugative mating reaction with <i>Agrobacterium tumefaciens</i> strain GV3101. In this assay, a donor culture of <i>E. coli</i> containing RP1 was grown in LB supplemented with kanamycin (75 ug/ml), and a recipient culture of <i>Agrobacterium tumefaciens</i> strain GV3101 was grown in LB. The cultures were harvested at OD ___, mixed together at different 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 rifampicin (??/ml). This allowed only for growth of <i>A. tumefaciens</i> cells expressing the kanamycin selection marker from the obtained RP1 plasmid and a chromosomal rifampicin selection marker. To control for the possibility of false-positives from spontaneous kanamycin and rifampicin 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 | + | RP1 conjugation between <i>A. tumefaciens</i> |
| − | 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 RP1 inside A. tumefaciens strain GV3101 (obtained from the mating assay above) in LB supplemented with kanamycin (75 ug/ml), and a recipient culture of A. tumefaciens strain LBA4404 in LB supplemented with streptomycin (2000 ug/ml). Cultures were harvested at OD ___, concentrated to OD ___ by centrifugation, mixed together, and incubated for ___ 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. Control reactions of donor and recipient cultures were plated on the same conditions to ensure the absence of spontaneous kanamycin and streptomycin resistant mutants. | + | To investigate the ability of RP1 to conjugate between <i>Agrobacterium</i> cells, we designed another conjugation similar to the one above. We grew a donor culture of RP1 inside <i>A. tumefaciens</i> strain GV3101 (obtained from the mating assay above) in LB supplemented with kanamycin (75 ug/ml), and a recipient culture of <i>A. tumefaciens</i>strain LBA4404 in LB supplemented with streptomycin (2000 ug/ml). Cultures were harvested at OD ___, concentrated to OD ___ by centrifugation, mixed together, and incubated for ___ 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. Control reactions of donor and recipient cultures were plated on the same conditions to ensure the absence of spontaneous kanamycin and streptomycin resistant mutants. |
| − | Due to limitations | + | Due to limitations of the <i>A. tumefaciens</i> strains available, we were not able to obtain a recipient strain with a unique selection marker on its chromosome. Instead, we had to use the streptomycin resistance marker on strain LBA4404’s Ti-plasmid. Since the Ti-plasmid is also conjugative, albeit at low frequencies outside of specific inducing conditions, we quantified the ‘backwards’ conjugation of the strain LBA4404’s Ti-plasmid into the RP1-containing donor cells to ensure that this alternate scenario giving rise to kanamycin-streptomycin resistant cells was not confounding the assay. |
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Results | Results | ||
| − | Results/discussion of conjugation assay of E.coli and GV3101: | + | Results/discussion of conjugation assay of <i>E.coli</i> and GV3101: |
| − | The plates with both selection markers (kan selects for RP1 plasmid and gent selects for recipient) showed growth from all mating reaction of different ratios of donor (E. coli) and recipient GV3101. 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 recipient or donor. 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. | + | The plates with both selection markers (kanamycin (kan) selects for RP1 plasmid and gentamycin (gent) selects for recipient) showed growth from all mating reaction of different ratios of donor (<i>E. coli</i>) 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 recipient or donor. Taken together, this indicates that <i>Agrobacterium</i> 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 in the double-selection marker plates indicated that RP1 should exist within some bacteria due to it, and none other plasmid or strain, having kan resistance, and only GV3101 strain should grow on the plate due to it, and none other plasmid or strain, having gent resistance. | Growth in the double-selection marker plates indicated that RP1 should exist within some bacteria due to it, and none other plasmid or strain, having kan resistance, and only GV3101 strain should grow on the plate due to it, and none other plasmid or strain, having gent resistance. | ||
| − | 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??]. The negative controls gave us indication that E. coli (donor) and GV3101 (recipient) did not develop any spontaneous mutations for kan and gent resistance respectively. These results indicate that RP1 must have | + | 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??]. The negative controls gave us indication that E. coli (donor) and GV3101 (recipient) did not develop any spontaneous mutations for kan and gent resistance respectively. These results indicate that RP1 must have transferred from <i>E. coli</i> into GV3101 during the mating reactions. |
Revision as of 21:45, 1 November 2017
Conjugation Background
To be an effective countermeasure against crown gall disease, our CRISPR system targeting the Ti-plasmid must be capable of spreading efficiently in Agrobacterium tumefacienspopulation. We investigated a natural bacterial conjugation process for this purpose. Bacterial conjugation is the process by which a plasmid is spread from a donor cell directly into a recipient cell (). 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.
Design
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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 Agrobacterium tumefaciens strain GV3101 was grown in LB. The cultures were harvested at OD ___, mixed together at different 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 rifampicin (??/ml). This allowed only for growth of A. tumefaciens cells expressing the kanamycin selection marker from the obtained RP1 plasmid and a chromosomal rifampicin selection marker. To control for the possibility of false-positives from spontaneous kanamycin and rifampicin 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 RP1 inside A. tumefaciens strain GV3101 (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 ___, concentrated to OD ___ by centrifugation, mixed together, and incubated for ___ 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. Control reactions of donor and recipient cultures were plated on the same conditions to ensure the absence of spontaneous kanamycin and streptomycin resistant mutants. Due to limitations of the A. tumefaciens strains available, we were not able to obtain a recipient strain with a unique selection marker on its chromosome. Instead, we had to use the streptomycin resistance marker on strain LBA4404’s Ti-plasmid. Since the Ti-plasmid is also conjugative, albeit at low frequencies outside of specific inducing conditions, we quantified the ‘backwards’ conjugation of the strain LBA4404’s Ti-plasmid into the RP1-containing donor cells to ensure that this alternate scenario giving rise to kanamycin-streptomycin resistant cells was not confounding the assay. Results 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 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 recipient or donor. 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 in the double-selection marker plates indicated that RP1 should exist within some bacteria due to it, and none other plasmid or strain, having kan resistance, and only GV3101 strain should grow on the plate due to it, and none other plasmid or strain, having gent resistance. 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??]. The negative controls gave us indication that E. coli (donor) and GV3101 (recipient) did not develop any spontaneous mutations for kan and gent resistance respectively. These results indicate that RP1 must have transferred from E. coli into GV3101 during the mating reactions. Results/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.
Figure 1 Cloning procedure for ietAS.
Conclution
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References
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