Difference between revisions of "Part:BBa K4294093"
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pTU2-A-RFP (p15A ori): This is the low-copy version of pTU2-A-RFP (colE1 ori) due to the substitution of colE1 ori with the p15A origin of replication. We used pTU2-A-RFP (p15A ori) as a level 2-Golden Gate acceptor vector too for building the LuxI/TetR expression system in sender cells, alongside with the LuxI-mNeonGreen/TetR (later LuxI-sfGFP/TetR as seen in our Engineering Success tab) expression system for our RBS library characterization in the same cells. We chose a low-copy number plasmid for the expression of our system in senders, because we wanted the strength of each RBS to be reflected in the expression levels of LuxI/OC6 and not be compensated by the (potentially high) replication rate of the construct carrying it. We also utilized said vector to assemble one of the receivers’ circuits too, the OL expression system, to discover how protein expression rates might depend on plasmid copy number. Since it is identical with pTU2-A-RFP (colE1 ori) in its entire sequence except for the origin of replication, this vector also has a Cm resistance gene and measures approximately 3kbp in size. Like pTU2-A-RFP (colE1 ori), pTU2-A-RFP (p15A ori) is an RFP-dropout plasmid backbone. [1]
 
pTU2-A-RFP (p15A ori): This is the low-copy version of pTU2-A-RFP (colE1 ori) due to the substitution of colE1 ori with the p15A origin of replication. We used pTU2-A-RFP (p15A ori) as a level 2-Golden Gate acceptor vector too for building the LuxI/TetR expression system in sender cells, alongside with the LuxI-mNeonGreen/TetR (later LuxI-sfGFP/TetR as seen in our Engineering Success tab) expression system for our RBS library characterization in the same cells. We chose a low-copy number plasmid for the expression of our system in senders, because we wanted the strength of each RBS to be reflected in the expression levels of LuxI/OC6 and not be compensated by the (potentially high) replication rate of the construct carrying it. We also utilized said vector to assemble one of the receivers’ circuits too, the OL expression system, to discover how protein expression rates might depend on plasmid copy number. Since it is identical with pTU2-A-RFP (colE1 ori) in its entire sequence except for the origin of replication, this vector also has a Cm resistance gene and measures approximately 3kbp in size. Like pTU2-A-RFP (colE1 ori), pTU2-A-RFP (p15A ori) is an RFP-dropout plasmid backbone. [1]
  
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[[File:Addgene-plasmid-74090-sequence-138311-map.png]]
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[[File:Ptu2-a-rfp-p15a-ori-map.png]]
  
 
''Plasmid Map''
 
''Plasmid Map''

Revision as of 15:37, 13 October 2022


pTU2-A-RFP (p15A origin)


Usage and Biology

pTU2-A-RFP (p15A ori): This is the low-copy version of pTU2-A-RFP (colE1 ori) due to the substitution of colE1 ori with the p15A origin of replication. We used pTU2-A-RFP (p15A ori) as a level 2-Golden Gate acceptor vector too for building the LuxI/TetR expression system in sender cells, alongside with the LuxI-mNeonGreen/TetR (later LuxI-sfGFP/TetR as seen in our Engineering Success tab) expression system for our RBS library characterization in the same cells. We chose a low-copy number plasmid for the expression of our system in senders, because we wanted the strength of each RBS to be reflected in the expression levels of LuxI/OC6 and not be compensated by the (potentially high) replication rate of the construct carrying it. We also utilized said vector to assemble one of the receivers’ circuits too, the OL expression system, to discover how protein expression rates might depend on plasmid copy number. Since it is identical with pTU2-A-RFP (colE1 ori) in its entire sequence except for the origin of replication, this vector also has a Cm resistance gene and measures approximately 3kbp in size. Like pTU2-A-RFP (colE1 ori), pTU2-A-RFP (p15A ori) is an RFP-dropout plasmid backbone. [1]

Ptu2-a-rfp-p15a-ori-map.png

Plasmid Map


Reference

[1] Addgene (Plasmid #74090) https://www.addgene.org/74090/

[2] EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology. Moore SJ, Lai HE, Kelwick RJ, Chee SM, Bell DJ, Polizzi KM, Freemont PS. ACS Synth Biol. 2016 May 2. 10.1021/acssynbio.6b00031 PubMed 27096716


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found at 107
    Plasmid lacks a suffix.
    Illegal XbaI site found at 140
    Illegal XbaI site found at 150
    Illegal PstI site found at 1259
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 107
    Illegal PstI site found at 1259
    Illegal NotI site found at 113
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 107
    Illegal XhoI site found at 2231
    Illegal XhoI site found at 3123
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 107
    Plasmid lacks a suffix.
    Illegal XbaI site found at 140
    Illegal XbaI site found at 150
    Illegal PstI site found at 1259
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 107
    Plasmid lacks a suffix.
    Illegal XbaI site found at 122
    Illegal XbaI site found at 140
    Illegal XbaI site found at 150
    Illegal PstI site found at 1259
    Illegal AgeI site found at 942
    Illegal AgeI site found at 1054
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI site found at 128
    Illegal BsaI.rc site found at 1253