Plasmid

Part:BBa_K2797013:Design

Designed by: Kyle Stanforth   Group: iGEM18_Newcastle   (2018-10-04)


High copy BioBrick assembly plasmid pSB1C3 with RFP internal standard

Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 989
    Illegal suffix found in sequence at 1
    Illegal EcoRI site found at 3052
    Illegal XbaI site found at 3067
    Illegal SpeI site found at 1905
    Illegal PstI site found at 1919
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 989
    Illegal EcoRI site found at 3052
    Illegal NheI site found at 1017
    Illegal NheI site found at 1040
    Illegal SpeI site found at 2
    Illegal SpeI site found at 1905
    Illegal PstI site found at 16
    Illegal PstI site found at 1919
    Illegal NotI site found at 9
    Illegal NotI site found at 995
    Illegal NotI site found at 1912
    Illegal NotI site found at 3058
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 989
    Illegal EcoRI site found at 3052
    Illegal XhoI site found at 2036
    Illegal XhoI site found at 2928
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 989
    Illegal suffix found in sequence at 2
    Illegal EcoRI site found at 3052
    Illegal XbaI site found at 3067
    Illegal SpeI site found at 1905
    Illegal PstI site found at 1919
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 989
    Illegal EcoRI site found at 3052
    Illegal XbaI site found at 1004
    Illegal XbaI site found at 3067
    Illegal SpeI site found at 2
    Illegal SpeI site found at 1905
    Illegal PstI site found at 16
    Illegal PstI site found at 1919
    Illegal AgeI site found at 1627
    Illegal AgeI site found at 1739
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal EcoRI site found at 989
    Illegal EcoRI site found at 3052
    Illegal XbaI site found at 1004
    Illegal XbaI site found at 3067
    Illegal SpeI site found at 2
    Illegal SpeI site found at 1905
    Illegal PstI site found at 16
    Illegal PstI site found at 1919


Design Notes

Figure 2. The K2797013 plasmid following simulated Gibson Assembly of the RFP internal standard sequence into the pSB1C3 vector backbone. As shown, the RFP internal standard inserts between the chloramphenicol resistance gene and the ORI. The RFP gene is shown in pink flanked on the left by promoter BBa_J23108 and RBS BBa_0032, and flanked on the right by double terminator BBa_B0015.

Test device pSB1C3 were linearized via 2-step PCR in a non-coding region between the ORI and chloramphenicol resistance gene, a region deemed suitable due to its distance from the multiple cloning site, and treated using DpnI to remove non-amplified DNA. Gibson ends were designed using NEBuilder and the RFP BioBrick - containing the; gibson ends, promoter (BBa_J23108), RBS (BBa_0032), RFP coding sequence and double terminator (BBa_B0015) - was synthesized by IDT. Using Gibson Assembly, the RFP BioBrick was cloned into the pSB1C3 vector (Figure 3). The subsequent assembled plasmids were transformed into E. coli DH5-alpha using heat shock, cultured in LB broth and subjected to fluorescence analysis.

Source

This part was derived from the pSB1C3 test device plasmids used in the iGEM 2018 InterLab study

References