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