Plasmid Isolation of S. epidermidis and S. aureus

- Pellet cells from 2-10 mL of overnight culture.
- Centrifuge for 1 minute at 13000 rpm. Discard supernatant.
- Wash cells in 1 mL 3% sodium chloride solution. Re-suspend cells in saline solution, but be careful of splashing of the re-suspended solution.
- Centrifuge for 1 min at 13000 rpm. Discard supernatant.
- Re-suspend cell pellet in 150 μL of Solution I.
- Add 10 μL of lyostaphin and incubate at 37 °C for 30 min.
- Add 300 μL of solution II. Invert 10 times. Incubate on ice for 5 min.
- Add 225 μL of solution III. Invert 10 times. Incubate on ice for 5 min.
- Centrifuge contents at 13000 rpm for 5 min. Transfer supernatant to new tube.
- Transfer supernatant to a spin column and incubate for 2 mins at room temperature.
- Centrifuge spin column at 13000 rpm for 30 s. Discard flow-through.
- Add 500 μL of wash solution. Centrifuge at 13000 rpm for 30 s. Discard flow-through.
- Repeat previous step
- If you are seeking especially pure DNA, for example if the DNA is to be used for sequencing, repeat previous step a second time.
- Centrifuge at 13000 rpm for 2 min to remove remaining wash solution, discard flow-through.
- Transfer spin column to a new microfuge tube.
- Add 30-50 μL (depending on the desired concentration) elution buffer. Incubate 2 min at room temperature. For higher concentration, you may incubate at 65 °C (use the 42 °C heat block).
- If the samples are to send to BioBasic for sequencing, elute in 40 μL of Milli-Q water.
- Centrifuge at 13000 rpm for 1 min to elute DNA. Discard spin column.

Note that the protocol was adapted from Staphylococcus Epidermidis: Methods and Protocols by Springer Protocols, edited by Paul D. Fey. The protocol provided was used phenol-chloroform extraction, but the phenol-extraction steps were replaced with a spin column.

Transformation Protocol of S. epidermidis/aureus

- Inoculate 10 mL TSB (+25 μL tetracycline if S. epidermidis) overnight culture (12-16 h).
- Dilute the overnight culture to an OD@578 nm of ~0.5 in fresh pre-warmed media. Note the volume (x).
- Incubate cultures for 30 min at 37 °C, then chill on ice for 20 min with all subsequent steps performed on ice (or 4 °C).
- Centrifuge culture down at 4000 x g for 10 min at 4 °C.
- Re-suspend cells in an equal volume (equal to the initial diluted volume, x) of ice cold water (Autoclaved Milli-Q is acceptable).
- Repeat previous centrifugation and re-suspension steps.
- Centrifuge and re-suspend the cells in 1/10 (x/10) , then 1/25 (x/25), then 1/200th (x/200) of initial dilution in pre-warmed media.
- Make 50 μL aliquots and store in -80 °C freezer.
- When electroporating, let the cells thaw on ice for 5 min, then left at room temperature for 5 min.
- Centrifuge for 5 min at 5000 g for 1 min.
- Re-suspend pellet in 50 μL of 10% glycerol +500 mM sucrose solution (filter sterilized).
- Mix DNA (5 μg) and cells, and let incubate for 20 min.
- Electroporate cells with 2.1 kV (21 kV/cm) in a 0.1 cm electroporation cuvette.
- Add 1 mL of TSB and gently re-suspend the cells into the media in the cuvette.
- Transfer to a test tube and incubate for 2 h on a shaker at 37 °C.
- Plate cells on TSA plate with the correct antibiotic concentration for ~2 days.

Monk, I.R. et al. “Transforming the Untransformable: Application of Direct Transformation to Manipulate Genetically Staphylococcus aureus and Staphylococcus epidermidis”.mBio. 3(2). 1-11. doi:10.1128/mBio.00277-11.

sarA P1 Promoter Efficacy Assay:

The sarA P1 promoter (BBa_K1323021) and a J23101 promoter were inserted in front of a GFP coding sequence (BBa_ E0240). The resulting two plasmid constructs (now a GFP cassette) were independently transformed into DH5α. A diagnostic restriction digest was run to confirm the insert composition within each of these two GFP cassettes. Colonies were streak purified and grown on LB supplemented with an appropriate antibiotic. Overnight cultures were then prepared in triplicate. 50 µL was then used to inoculate fresh media. When each media preparation reached an OD600 of 0.5-0.6, the GFP fluorescence was measured on a plate reader. Quantification of sarA P1 is therefore measured relative to the amount of YFP produced from the standard J23101 promoter.

sRNAi Silencing Assay:

The YFP (BBa_K1323010) cassette was co-transformed with two sRNA constructs (BBa_K1323006, BBa_K1323007) into DH5α. Transformation of a YFP cassette on it’s own served as a positive control for YFP expression, while an sRNA cassette (BBa_K1323008) which targets a non-YFP sequence was the negative control. Colonies were streak purified and grown in LB supplemented with ampicillin. A diagnostic restriction digest was run to confirm that co-transformation was successful in the three co-transformed colonies. The digests were all done with EcoR1 and Pst1; in this way the inserts of each of the two co-transformed plasmids could be distinguished by gel electrophoresis. Overnight cultures were then prepared in triplicates. 50 µL of overnight culture was used to inoculate fresh media. When each media preparation reached an OD600 of 0.5-0.6, the YFP fluorescence was measured on a plate reader.

Antibiotic Resistance Profile

The construct containing both chloramphenicol and erythromycin (pSB1CE3 shuttle vector) was electroporated into S. epidermidis. Three colonies were streak purified and diagnostic restriction digests were done to confirm that the insert was present. Overnight TSB cultures were supplemented with 10 μg/mL erythromycin, and the inoculation was done in triplicate. The negative control for this experiment is a stock S. epidermidis (ATCC12228). It lacks erythromycin resistance but has chloramphenicol resistance (S. epiderimidis has this resistance endogenously). 10 μL of the overnight cultures were used to inoculate fresh TSB that was supplemented with 10, 50, or 100 μg/mL of erythromycin and chloramphenicol. The negative growth control was 75 μg/mL of kanamycin and the positive growth control was TSB without antibiotic. The samples were incubated at 37 ͒°C shaking at 200 rpm. The OD600 was read after 6 hours of incubation.

YFP expression over time:

The expression cassette (in the pSB3K3 backbone) contains the YFP coding sequence under a constitutive sarA promoter (BBa_K1323021) and TIR ribosome binding site (BBa_K1323016). DH5α either containing the YFP plasmid or containing no plasmids was grown in triplicate overnight at 37 °C in LB supplemented with the appropriate antibiotics. Cultures were diluted 1/100 into fresh LB with appropriate antibiotics and incubated at 37 °C. Every hour, the fluorescence (excitation: 485 nm, emission: 535 nm) and optical density at 600 nm of 200 μL of culture was read using a plate reader.