Difference between revisions of "Part:BBa K4294092"

Latest revision as of 15:41, 13 October 2022

pTU2-A-RFP (colE1 origin)

Plasmid Vector from the EcoFlex Modular MoClo kit.

Usage and Biology

We used pTU2-A-RFP (colE1 ori) as a level 2 (as indicated by the “2” in its name) - Golden Gate acceptor vector for assembling all different versions of the mNeonGreen/LuxR expression system in receivers: Open Loop (OL or OpLo), Positive Feedback (PF), Positive Feedback with a constitutive promoter (PFc), Positive Feedback with a Repressor (PF+R) and Positive Feedback with a Repressor and a constitutive promoter (PFc+R). This vector has a chloramphenicol (Cm) resistance gene and is a medium copy plasmid; it is approximately 3kbp in size. We selected a medium-copy plasmid for the expression of our system in receiver cells, as such a plasmid would allow the production of our visual output (mNeonGreen) while maintaining it at the levels receivers are actually activated by OC6 molecules; namely, without having a potentially high-copy vector counterbalance the different amount of AHL molecules receivers are stimulated by. This medium-copy backbone would not stress out cells carrying it either. Another reason we chose this particular vector was its marker; pTU2-A-RFP (colE1 ori) contains an RFP gene, enabling screening of colonies expressing recombinant plasmids without the need to use additional reagents (magenta colonies: transformed with the undesired parental plasmid, white colonies: transformed with a recombinant plasmid). [1] [2]

Plasmid Map

Reference

[1] Addgene plasmid # 74093 ; http://n2t.net/addgene:74093 ; RRID:Addgene_74093

[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

@@ Line 7: / Line 7: @@
 ===Usage and Biology===
-We used pTU2-A-RFP (colE1 ori) as a level 2 (as indicated by the “2” in its name) - Golden Gate acceptor vector for assembling all different versions of the mNeonGreen/LuxR expression system in receivers: Open Loop (OL or OpLo), Positive Feedback (PF), Positive Feedback with a constitutive promoter (PFc), Positive Feedback with a Repressor (PF+R) and Positive Feedback with a Repressor and a constitutive promoter (PFc+R). This vector has a chloramphenicol (Cm) resistance gene and is a medium copy plasmid; it is approximately 3kbp in size. We selected a medium-copy plasmid for the expression of our system in receiver cells, as such a plasmid would allow the production of our visual output (mNeonGreen) while maintaining it at the levels receivers are actually activated by OC6 molecules; namely, without having a potentially high-copy vector counterbalance the different amount of AHL molecules receivers are stimulated by. This medium-copy backbone would not stress out cells carrying it either.  Another reason we chose this particular vector was its marker; pTU2-A-RFP (colE1 ori) contains an RFP gene, enabling screening of colonies expressing recombinant plasmids without the need to use additional reagents (magenta colonies: transformed with the undesired parental plasmid, white colonies: transformed with a recombinant plasmid).
+We used pTU2-A-RFP (colE1 ori) as a level 2 (as indicated by the “2” in its name) - Golden Gate acceptor vector for assembling all different versions of the mNeonGreen/LuxR expression system in receivers: Open Loop (OL or OpLo), Positive Feedback (PF), Positive Feedback with a constitutive promoter (PFc), Positive Feedback with a Repressor (PF+R) and Positive Feedback with a Repressor and a constitutive promoter (PFc+R). This vector has a chloramphenicol (Cm) resistance gene and is a medium copy plasmid; it is approximately 3kbp in size. We selected a medium-copy plasmid for the expression of our system in receiver cells, as such a plasmid would allow the production of our visual output (mNeonGreen) while maintaining it at the levels receivers are actually activated by OC6 molecules; namely, without having a potentially high-copy vector counterbalance the different amount of AHL molecules receivers are stimulated by. This medium-copy backbone would not stress out cells carrying it either.  Another reason we chose this particular vector was its marker; pTU2-A-RFP (colE1 ori) contains an RFP gene, enabling screening of colonies expressing recombinant plasmids without the need to use additional reagents (magenta colonies: transformed with the undesired parental plasmid, white colonies: transformed with a recombinant plasmid). [1] [2]
@@ Line 17: / Line 17: @@
 [1] Addgene plasmid # 74093 ; http://n2t.net/addgene:74093 ; RRID:Addgene_74093
 [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