Difference between revisions of "Team:British Columbia/Growing"

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 was G. apicola or S. alvi.

Figure 3: G. apicola in TSB.

Figure 4: G. apicola in TSB with oxytetracycline.

Table 1: Types of liquid media tested for growth of S. alvi and G. apicola.

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 it’s slow growth, it takes approximately 24 hours to reach a stationary-like growth phase.

Figure 5: G. apicola growth curve.

Two protocols were attempted for the creation of electrocompetent cells (protocols): one designed for Campylobacter jejuni (similar to G. apicola due to it's microaerophilicity) and one designed for Salmonella (similar to G. apicola, a γ-proteobacterium).

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 designed to create chemically competent cells of E. coli was attempted (protocol), due to it's cladistic similarity to G. apicola. 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 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 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 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.

“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.