Plasmid_Backbone

Part:BBa_K1323017

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

E. coli - Staphylococcus Shuttle Vector

This part is an improvement of the pSB1C3 backbone. This improvement makes the backbone much more versatile by turning into a functional E. coli - Staphylococcus shuttle vector. The Staphylococcus oriV from the pSK41 plasmid BBa_K1323018 and an erythromycin resistance gene BBa_K1323011 was inserted between the VR and rep (pMB1) regions in pSB1C3 using BamHI and BglII restriction sites. The part contains an RFP cassette BBa_J04450 between the prefix and suffix.


Resistances Graph2.png



The construct (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 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
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found at 4855
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 4855
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal NotI site found at 9
    Illegal NotI site found at 4861
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 4855
    Illegal BglII site found at 2978
    Illegal BamHI site found at 178
    Illegal BamHI site found at 186
    Illegal XhoI site found at 3839
    Illegal XhoI site found at 4731
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 4855
    Illegal suffix found at 2
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 4855
    Plasmid lacks a suffix.
    Illegal XbaI site found at 4870
    Illegal SpeI site found at 2
    Illegal PstI site found at 16
    Illegal AgeI site found at 5657
    Illegal AgeI site found at 5769
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 4855
    Illegal XbaI site found at 4870
    Illegal SpeI site found at 2
    Illegal PstI site found at 16


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