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Part:BBa_K1323011

Designed by: Julia Manalil   Group: iGEM14_Waterloo   (2014-10-04)

Erythromycin resistance coding sequence

This coding sequence is for the antibiotic, erythromycin. This part is PCR amplified from ermM cassette synthesized by Bio Basic. 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 µg/mL in S.epidermidis/S.aureus hosts.


Resistances Graph.png

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 µg/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 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.



Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

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.


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Categories
//cds/selectionmarker/antibioticresistance
Parameters
None