Plasmid Isolation of S. epidermidis and S. aureus

- Pellet cells from 2-10mL of overnight culture.
- Centrifuge for 1 minute at 13000rpm. Discard supernatant.
- Wash cells in 1mL 3% sodium chloride solution. Resuspend cells in saline solution, but be careful of splashing of the resuspended solution.
- Centrifuge for 1min at 13000rpm. Discard supernatant.
- Resuspend cell pellet in 150uL of Solution I.
- Add 10uL of lyostaphin and incubate at 37 C for 30mins.
- Add 300uL of solution II. Invert 10 times. Incubate on ice for 5mins.
- Add 225uL of solution III. Invert 10 times. Incubate on ice for 5mins.
- Centrifuge contents at 13000 rpm for 5mins. 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 seconds. Discard flow-through.
-Add 500 ul of wash solution. Centrifuge at 13000 rpm for 30 seconds. 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 30-50uL (depends on the concentration wanted)elution buffer. Incubate 2 mins at room temperature. For higher concentration, you may incubate at 65 degrees Celsius (use the 42 degree Celsius heat block)
- If the samples are to send to Biobasic for sequencing, elute in 40uL of MilliQ 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

- Inocculate 10mL TSB (+25uL tetracycline if S. epidermidis) overnight culture (12-16 hours)
- Dilute the overnight culture to an OD@578nm of ~0.5 in fresh prewarmed media, note the volume (x)
- Incubate cultures for 30mins at 37 deg C and then chill on ice for 20mins with all subsequent steps performed on ice (or 4 deg C)
- Centrifuge culture down at 4000xg for 10min at 4 deg C
- Resuspend cells in an equal volume (equal to the initial diluted volume, x) of ice cold water (Autoclaved MiliQ is fine)
- Repeat previous centrifugation and resuspension steps
- Centrifuge and resuspend the cells in 1/10 (x/10) , then 1/25 (x/25), then 1/200th (x/200) of initial dilution in prewarmed media.
- Make 50uL aliquots and store in -80 degree freezer
- When electroporating, let the cells thaw on ice for 5mins then left at room temperature for 5mins
- Centrifuge for 5mins at 5000g for 1min
- Resuspend pellet in 50uL of 10%glycerol +500mM sucrose solution (filter sterilized)
- Mixed DNA (5ug) and cells and let incubate for 20mins.
- Electroporate cells with 2.1kV (21kV/cm) in a 0.1cm electroporation cuvette
- Add 1mL of TSB and gently resuspend the cells into the media in the cuvette
- Transfer to a test tube and incubate for 2 hours on a shaker at 37 deg 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 ug/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 uL of the overnight cultures were used to inoculate fresh TSB that was supplemented with 10, 50, or 100 ug/mL of erythromycin and chloramphenicol. The negative growth control was 75 ug/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.