Difference between revisions of "Part:BBa K1323011"

Revision as of 01:06, 17 October 2014

Erythromycin resistance coding sequence

This coding sequence is for the antibiotic, erythromycin. This part is PCR amplified from ermM cassette synthesized by Biobasic. This ermM cassette is originally found in S.epidermidis plasmid pNE131 which confers resistance to macrolide-lincosamide-streptogramin B group antibiotics including erythromycin (Catchpole et al., 1988). Working concentration for this antibiotic is 10 ug/mL in S.epidermidis/S.aureus hosts.

• Add antibiotic resistance profile graph (dropbox/lab&design/results)

The construct (erythromycin cds in pUC57) 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 was S.epidermidis (ATCC12228), it lacks erythromycin resistance but has chloramphenicol resistance. 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.

Catchpole, I., Davies, T. A., and Dyke, K. G. H. (1988). The nucleotide sequence of Staphylococcus aureus plasmid pT48 conferring inducible macrolide-lincosamide-streptogramin B resistance and comparison with similar plasmids expressing constitutive resistance. Journal of General Microbiology 134, 697-709.

Sequence and Features