Difference between revisions of "Team:British Columbia/Growing"
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| − | <p>Transformation of <i>G. apicola</i> was tested using electroporation (2). Click here to view the protocol. The transformed bacteria were plated on TSA to recover overnight to allow for expression of antibiotic resistance genes or recovered in anaerobic TSB for 1.5 hours. Cells were then transferred onto the appropriate antibiotic plates, supplemented with oxytetracycline (30 μg/mL) added to further select for <i>G. apicola</i> due to its natural resistance. Plates were incubated at 37°C under microaerophilic conditions for 24-48 hours. | + | <p>Transformation of <i>G. apicola</i> was tested using electroporation (2). <a href="https://static.igem.org/mediawiki/2015/1/1d/UBC_electroporationprotocol.pdf">Click here</a> to view the protocol. The transformed bacteria were plated on TSA to recover overnight to allow for expression of antibiotic resistance genes or recovered in anaerobic TSB for 1.5 hours. Cells were then transferred onto the appropriate antibiotic plates, supplemented with oxytetracycline (30 μg/mL) added to further select for <i>G. apicola</i> due to its natural resistance. Plates were incubated at 37°C under microaerophilic conditions for 24-48 hours. |
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| − | <p>Following a standard protocol for heat shocking <i>E. coli</i>, transformation with each plasmid was tested with competent <i>G. apicola</i>. Click here to view the protocol. Cells were recovered on a TSA plate for 24 hrs (or in anaerobic TSB for 1 hour) before biomass was harvested and a portion of the recovered cells were plated on the appropriate antibiotic plate for selection of transformants. | + | <p>Following a standard protocol for heat shocking <i>E. coli</i>, transformation with each plasmid was tested with competent <i>G. apicola</i>. <a href="https://static.igem.org/mediawiki/2015/7/71/UBC_heatshockprotocol.pdf">Click here</a> to view the protocol. Cells were recovered on a TSA plate for 24 hrs (or in anaerobic TSB for 1 hour) before biomass was harvested and a portion of the recovered cells were plated on the appropriate antibiotic plate for selection of transformants. |
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| − | <p>Following a modified protocol that can be viewed here, <i>G. apicola</i> was grown on a TSA plate for 48 hrs. Concurrently, a conjugative <i>E.coli</i> strain (SM10 or S17) harboring the desired plasmid to be mobilized was grown in 5 mL LB and antibiotic for 24 hrs. <i>G. apicola</i> (scraped off a TSA plate) and E. coli were combined and pelleted together, resuspended in 100 μL of LB, plated on TSA, and incubated in a microaerophilic environment for 24 hrs at 37C. Colonies were then replated on selective plates containing antibiotic specific to the plasmid used and oxytetracycline (30μg/mL) to select for <i>G. apicola</i>. <i>G. apicola</i> is naturally resistant to oxytetracycline(1). | + | <p>Following a modified protocol that can be viewed <a href="https://static.igem.org/mediawiki/2015/b/b6/UBC_conjugationprotocol.pdf">here</a>, <i>G. apicola</i> was grown on a TSA plate for 48 hrs. Concurrently, a conjugative <i>E.coli</i> strain (SM10 or S17) harboring the desired plasmid to be mobilized was grown in 5 mL LB and antibiotic for 24 hrs. <i>G. apicola</i> (scraped off a TSA plate) and E. coli were combined and pelleted together, resuspended in 100 μL of LB, plated on TSA, and incubated in a microaerophilic environment for 24 hrs at 37C. Colonies were then replated on selective plates containing antibiotic specific to the plasmid used and oxytetracycline (30μg/mL) to select for <i>G. apicola</i>. <i>G. apicola</i> is naturally resistant to oxytetracycline(1). |
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Revision as of 01:53, 18 September 2015
Genetic Tool Development
For the probiotic, the β-proteobacteria, Snodgrassella alvi, and the γ-proteobacteria, Gilliamella apicola, were chosen since it is distinctly endogenous to the midgut of the European honey bee, Apis mellifera (1). Since these microaerophiles are native and unique to the honey bee gut, introducing imidacloprid and 6-CNA degradation genes into these candidate bacteria would minimize the chance of resistance genes spreading to other insects. Due to the limited amount of existing literature on G. apicola and S. alvi, the project focused on discovering methods to make these bacteria genetically tractable. This included culturing the bacteria on different growth mediums, testing methods to induce competence, and transformation techniques with a variety of plasmids.
Culturing
Due to the novel nature of using G. apicola and S. alvi for the project (as opposed to E. coli), the first step was to determine the optimal method of culturing either bacteria.
G. apicola and S. alvi were streaked on TSA, LB, and blood agar (5% sheep) plates and stored and grown at 37°C in an anaerobic jar flushed with 5% CO2 balanced with N2. Growth was tested on a variety of media types in order to determine the best growing conditions. Based on the visual indication of colony size, number, and growing time, G. apicola grew the best on TSA plates, while S. alvi grew best on blood agar (5% sheep blood). G. apicola and S. alvi colonies gave distinct colonies after 48 and 96 hours, respectively, in the microaerophilic chamber.
Figure 1: S. alvi on a 5% sheep blood agar plate. Figure 2: G. apicola on a TSA plate. As no liquid growth medium for S. alvi or G. apicola have been reported in literature, a variety of liquid media growth conditions were tested (see Table 1). All liquid cultures were incubated at 37°C for 72 hours, or until a turbidity was visibly detected. G. apicola grew in TSB with an anaerobic environment. Whilst growing in liquid media, it was noted that G. apicola aggregated into dense snowflake-like colonies (Figure 3). S. alvi was not successfully cultivated in any liquid media tested. Colony PCR and plating on oxytetracycline plates under microaerophilic conditions were used to confirm growth was G. apicola or S. alvi.
Figure 1: S. alvi on a 5% sheep blood agar plate. Figure 2: G. apicola on a TSA plate. | Liquid Media | Method | S. alvi | G. apicola |
|---|---|---|---|
| TSB | Flushed with 5% CO2 Balanced with N2 while cold | No Growth | No Growth |
| LB | Flushed with 5% CO2 Balanced with N2 while cold | No Growth | No Growth |
| SOC | Flushed with 5% CO2 Balanced with N2 while cold | No Growth | No Growth |
| MH Broth | Flushed with 5% CO2 Balanced with N2 while cold | No Growth | No Growth |
| Brain Heart Infusion | Flushed with 5% CO2 Balanced with N2 while cold | No Growth | No Growth |
| TSB | Flushed with 5% CO2 Balanced with N2 while hot | No Growth | Successful Growth |
Table 1: Types of liquid media tested for growth of S. alvi and G. apicola.
Growth Curve
The growth curve of G. apicola was monitored on a plate reader that measured the OD value at 600nm over 36 hours. G. apicola was inoculated into a TSB culture that was previously flushed with 5% CO2 balanced with N2. Additionally, 5% CO2 balanced with N2 was blown onto the plate whilst sealing to ensure the lowest possible amount of oxygen was present in the plate.
The growth curve of G. apicola in a TSB culture was monitored on a plate reader that took OD values at 600nm. As displayed in figure 3 G. apicola’s lag phase lasts 15 hours. Moreover, due to it’s slow growth, it takes approximately 24 hours to reach a stationary-like growth phase.
Figure 1: S. alvi on a 5% sheep blood agar plate. Inducing Competence in G.apicola and S.alvi
After identifying the optimal way to culture G. apicola, we moved on to attempting various ways of inducing competence in the bacteria. Due to no existing literature on methods of inserting a plasmid into G. apicola, various protocols known to work on other gram-negative gammaproteobacteria, as well as a protocol for microaerophilic bacteria were attempted. View our protocols here, under Genetic Tool Development.
Two protocols were attempted for creating electrocompetent cells (protocols): one designed for Campylobacter jejuni (similar to G. apicola because it is a microaerophilic bacteria) and one designed for Salmonella (similar to G. apicola because it is also a γ-proteobacteria).
Following the protocol from Methods in Microbiology: Bacterial Pathogenesis for Campylobacter jejuni by Williams, P., Ketley, J., & Salmond, G. (2), G. apicola was grown on TSA for 48 hours at 37°C, after which the biomass was removed. Cells were washed with ice cold wash buffer of sucrose and glycerol. Competent cells were then stored at -80°C, or transformed immediately by electroporation.
For the second method, the protocol from Methods in Microbiology, Vol. 47: Electroporation Protocols for Microorganisms (Salmonella) by Nickoloff, J. A. (3), was used to induce competence in G. apicola after 48 hrs of growth at 37°C. Biomass was harvested and washed with HEPES buffer and 10% glycerol. Competent cells were either stored at -80°C or transformed immediately by electroporation.
One protocol for creating chemically competent cells was attempted (protocol): designed for E. coli (similar to G. apicola because it is also a γ-proteobacteria). G. apicola was grown on TSA for 48 hours at 37C, after which the biomass was removed. Cells were then washed with a CaCl2 buffer. Competent cells were stored at -80C, or transformed immediately by heat shock.
Transformation
After creating the competent cells, we attempted a variety of transformation protocols. View our protocols here, under Genetic Tool Development.
Transformation of G. apicola was tested using electroporation (2). Click here to view the protocol. The transformed bacteria were plated on TSA to recover overnight to allow for expression of antibiotic resistance genes or recovered in anaerobic TSB for 1.5 hours. Cells were then transferred onto the appropriate antibiotic plates, supplemented with oxytetracycline (30 μg/mL) added to further select for G. apicola due to its natural resistance. Plates were incubated at 37°C under microaerophilic conditions for 24-48 hours.
Following a standard protocol for heat shocking E. coli, transformation with each plasmid was tested with competent G. apicola. Click here to view the protocol. Cells were recovered on a TSA plate for 24 hrs (or in anaerobic TSB for 1 hour) before biomass was harvested and a portion of the recovered cells were plated on the appropriate antibiotic plate for selection of transformants.
Following a modified protocol that can be viewed here, G. apicola was grown on a TSA plate for 48 hrs. Concurrently, a conjugative E.coli strain (SM10 or S17) harboring the desired plasmid to be mobilized was grown in 5 mL LB and antibiotic for 24 hrs. G. apicola (scraped off a TSA plate) and E. coli were combined and pelleted together, resuspended in 100 μL of LB, plated on TSA, and incubated in a microaerophilic environment for 24 hrs at 37C. Colonies were then replated on selective plates containing antibiotic specific to the plasmid used and oxytetracycline (30μg/mL) to select for G. apicola. G. apicola is naturally resistant to oxytetracycline(1).
Colonies forming on antibiotic plates were subject to two PCRs to confirm identity as G. apicola and for the presence of transformed plasmid. To confirm identity, a portion of the G. apicola 16S ribosomal subunit was amplified using specific primers(5). Length and sequence were confirmed by DNA agarose gel electrophoresis and sequencing. Additionally, the colonies were streaked out onto another antibiotic plate to ensure single colony morphology and stability of the plasmid. A second PCR was done to confirm presence of a plasmid. Primers specific for the plasmid were used to amplify a portion of the plasmid and confirmed by DNA agarose gel electrophoresis. As a positive control, all protocols for inducing competence were tested on E. coli DH5α with plasmid pSB1A3. Selection of antibiotic and PCR were used to confirm that transformed E. coli were harboring the plasmid used. Competence was successful induced in a model gram-negative γ-proteobacteria, E. coli. Table 2 summarizes the variety of methods tested and results obtained. Unfortunately, there were no replicated successes.
| Plasmid Name | Antibiotic Resistance Cassette | E. coli Heat Shock Protocol | C. jejuni Electroporation Protocol | S. enteritidis Electroporation Protocol | Conjugation |
|---|---|---|---|---|---|
| pSB1C3 | Chloramphenicol | No growth | No growth | NA | Not mobilizable/ conjugative |
| pSB1A3 | Ampicillin | No growth | No growth | No growth | Not mobilizable/ conjugative |
| pBBR1MCS-2 | Kanamycin | No growth | No growth | No growth | Not mobilizable/ conjugative |
| RP1 | Kanamycin, Ampicillin, Tetracycline | No growth | No growth | NA | No growth |
| pKT210 | Streptomycin | NA | No growth | No growth | No growth |
| pRK293 | Kanamycin | NA | No growth | NA | No growth |
| pIND4 | Kanamycin | No growth | No growth | NA | No growth |
| pBBR1MCS-3 | Tetracycline | NA | No growth | No growth | Not mobilizable/ conjugative |
| pBSPIISK- | Ampicillin | NA | No growth | NA | No growth |
| pBBR 3 | Steptomycin | NA | No growth | NA | Not mobilizable/ conjugative |
“NA” indicates a plasmid and inducing competence protocol mix that was unable to be performed due to time and material restraints.
Table 2: Summary of methods tested to induce competence and plasmids subsequently tested for transformation in G. apicola.
Acknowledgements
We would like to thank the following people greatly for their assistance, suggestions, and providing the plasmids/materials for us to experiment with.
Walden Kwong for providing the strains of G. apicola and S. alvi.
Dr. Julian Davies for providing the RP1 plasmid.
Dr. John Smit and Dr. John Nomellini for providing the E.coli S17, and SM-10 strains. As well for providing the plasmids PBBR3, PBBR4, PKT210, and PRK293.
Dr. Rachel Fernandez for providing the PBBRMCS1-2 plasmid.
Dr. J. Thomas Beatty for providing the PIND4 plasmid.
Dr. Bob Hancock and Dr. Mangeet Bains for providing PBBR1MCS-3, PBBR1MCS-5, and PBSPIISK(-).
Dr. Michael Murphy and everyone in the Murphy Lab for being amazing hosts.
References
- Kwong, W., Engel, P., Koch, H., and Moran, N. (2014). Genomics and host specialization of honey bee and bumble bee gut symbionts. Proceedings of the National Academy of Sciences, 111, 11509-11514.
- Williams, P., Ketley, J., & Salmond, G. (Eds.). (1998). Bacterial Pathogenesis. London, UK: Academic Press.
- Nickoloff, J. A. (Ed.). (1995). Electroporation Protocol for Microorganisms. Totowa, NJ: Humana Press Inc.
- Van der Geize, R. et al. (2002). Molecular and functional characterization of kshA and kshB, encoding two components of 3-ketosteroid 9α-hydroxylase, a class IA monooxygenase, in Rhodococcus erythropolis strain SQ1. Molecular Microbiology, 45(4). doi:0.1046/j.1365-2958.2002.03069
- Koch, H. et al. (2013). Diversity and evolutionary patterns of bacterial gut associates of corbiculate bees. Molecular Ecology, 22(7). doi: 10.1111/mec.12209
- Cho, J. et al. (2003). The Effects of Altering Autoinducer-2 Concentration on Transfer Efficiencies of the F and RPI plasmids to the Quorum Sensing Recicpient Escherichia coli Strain AB1157. Journal of Experimental Microbiology and Immunology (JEMI), 3, pp. 8-14.
- Chan, V. et al. (2002). The Effect of Increasing Plasmid Size on Transformation Efficiency in Escherichia coli. Journal of Experimental Microbiology and Immunology (JEMI), 2, pp. 207-223.
- Rodriguez, R. L, & Denhardt, D. T. (1988). Vectors: A Survey of Molecular Cloning Vectors and Their Uses. Stoneham, MA: Butterworth Publishers.
- Plasmid map of pIND4 for Rhodobacter sphaeroides. (2005). Retrieved August 5, 2015.
- Schweizer, H. P. (2001). Vectors to express foreign genes and techniques to monitor gene expression in Pseudomonads. Curr. Opin. Biotechnol. 12:439–445.
- Kovach, M.E., Elzer, P.H., Steven Hill, D., Robertson, G.T., Farris, M.A., Roop, R.M., and Peterson, K.M. (1995). Four New Derivatives of the Broad-Host-Range Cloning Vector pBBR1MCS, Carrying Different Antibiotic-Resistance Cassettes. Gene, 166(1). 175-176.
UBC iGEM 2015