Team:British Columbia/Growing
Genetic Tool Development
The γ-proteobacteria, Gilliamella apicola, and the β-proteobacteria, Snodgrassella alvi, were chosen as candidates for our probeeotic due to their endogenous nature in relation to the midgut of the European honeybee, Apis mellifera (1). Native and unique to the honeybee gut, the introduction of 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 media, testing methods of competence induction, and transformation techniques with a variety of plasmids.
Culturing
Due to the novelty of using G. apicola and S. alvi for the project (vs. E. coli), the first step was to identify the optimal method of culturing either bacteria.
G. apicola and S. alvi were streaked on TSA, LB, and blood agar (5% sheep blood) 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 to identify the best growing conditions. Based on the visual estimations 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 turbidity could be visually detected. G. apicola grew in anaerobic TSB. During growth in liquid media, it was noted that G. apicola aggregated into dense snowflake-like colonies (Figure 3). S. alvi did not grow in any liquid media tested. Colony PCR and plating on oxytetracycline plates under microaerophilic conditions were used to confirm growth as G. apicola or S. alvi.
Figure 3: G. apicola in TSB. 
Figure 4: G. apicola in TSB with oxytetracycline. | 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 of G. apicola was monitored on a plate reader that measured the OD value at 600nm over 36 hours, and plotted to a curve at fixed time points. For this, 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 presence of a minimal amount of oxygen 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 5, G. apicola’s lag phase lasts 15 hours. Moreover, due to its slow growth, it takes approximately 24 hours to reach a stationary-like growth phase.
Figure 5: G. apicola growth curve. Inducing Competence in G.apicola and S.alvi
After identifying the optimal method to culture G. apicola, we moved on to attempting various ways of inducing competence in the bacteria. Due to the lack of existing literature on methods of inserting a plasmid into G. apicola, we tried various protocols known to work on other gram-negative gammaproteobacteria, and a protocol for microaerophilic bacteria were attempted. View our protocols here, under Genetic Tool Development.
Three protocols were attempted for the creation of electrocompetent cells (protocols): one designed for Campylobacter jejuni (similar to G. apicola due to its microaerophilicity), one designed for Salmonella (similar to G. apicola, a γ-proteobacterium), and the last one designed as a general procedure for inducing electrocompetence.
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.
For the last method, a general electrocompetence procedure was used to induce electrocompetence in G. apicola after 24-30 hours of growth in liquid TSB at 37°C. Bacteria was pelleted with a microcentrifuge and re-suspended in decreasing volumes of sterile deionized water several times, with the last re-suspension being in sterile deionized water + 20% glycerol. The bacteria were then aliquoted (60 μL) into 1.7 mL Eppendorf tubes and subjected to snap freezing with liquid nitrogen. The now-competent cells were either stored at -80°C or transformed immediately by electroporation.
One protocol designed to create chemically competent cells of E. coli was attempted (protocol), due to its cladistic similarity to G. apicola. G. apicola was grown on TSA for 48 hours at 37°C, after which the biomass was removed. Cells were then washed with a CaCl2 buffer. Competent cells were stored at -80°C, or transformed immediately by heat shock.
Transformation
After creating the competent cells, a variety of transformation protocols were attempted. 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 the transformation of E. coli via heat shock, each procured plasmid was used in attempts to transform 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) after which 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) harbouring 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 hours at 37°C. 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 PCR reactions:one to confirm identity as G. apicola and another to test for the presence of transformed plasmid. To confirm bacterial 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 (13) | Kanamycin | No growth | No growth | No growth | Not mobilizable/ conjugative |
| RP1 (7) | Kanamycin, Ampicillin, Tetracycline | No growth | No growth | NA | No growth |
| pKT210 (8) | Streptomycin | NA | No growth | No growth | No growth |
| pRK293 (9) | Kanamycin | NA | No growth | NA | No growth |
| pIND4 (10) | Kanamycin | No growth | No growth | NA | No growth |
| pBBR1MCS-3 (13) | Tetracycline | NA | No growth | No growth | Not mobilizable/ conjugative |
| pBSPIISK- (11) | Ampicillin | NA | No growth | NA | No growth |
| pBBR 3 (13) | 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.
Waldan 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
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- Plasmid map of pIND4 for Rhodobacter sphaeroides. (2005). Retrieved August 5, 2015.
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UBC iGEM 2015