Segall-Shapiro TH, Meyer AJ, Ellington AD, Sontag ED, Voigt CA
Mol Syst Biol. 2014 Jul 30;10(7):742. PubMed Article
Christopher Voigt's lab has built a ‘resource allocator’ to control synthetic genetic systems by splitting T7 RNA polymerase (RNAP) variants into a number of fragments. By tuning the production of a limiting protein fragment, the total number of polymerases in a synthetic system can be controlled while allowing the allocation of this resource between different outputs.
This publication describes the bisection mapping of T7 RNAP to produce functional protein fragments and the composition of these fragments into a resource allocator system. Four orthogonal T7 RNAP variants were bisected into a conserved ‘core fragment’ and variable ‘σ fragments’. T7 RNAP is further shown to tolerate fragmentation into three and four fragments while retaining function. The three-piece RNAP and further mutations are used to create accessory fragments for further regulation of the resource allocator.
The Voigt lab has deposited plasmids encoding the fragmented T7 RNAP system that can be used to implement resource allocation. These include orthogonal T7 RNAP variant reporter plasmids (pTHSSd_8 – 11), plasmids with inducible σ fragments (pTHSSd_23 – 26), core fragment expression plasmids (pTHSSd_38 – 40), ‘accessory’ fragments (pTHSSd_27, 29, 42), and control plasmids (pTHSSd_34, 41, 44). They have also deposited plasmids to express three- and four-piece T7 RNAP (pTHSSd_14, 18) and a modified transposon that can be used to map the tolerated bisection sites in a protein (pTHSSd_1).
Plasmids from Article
| ID | Plasmid | Purpose |
|---|---|---|
| 59947 | pTHSSd_1 | Splitposon in KanR ColE1 backbone |
| 59948 | pTHSSd_8 | PT7 GFP reporter |
| 59949 | pTHSSd_9 | PT3 GFP reporter |
| 59950 | pTHSSd_10 | PK1F GFP reporter |
| 59951 | pTHSSd_11 | PCGG GFP reporter |
| 59952 | pTHSSd_14 | Triple split (at 67, 601-SZ) |
| 59953 | pTHSSd_18 | Quad split (at 61, 179, 601-SZ) |
| 59954 | pTHSSd_23 | PTac T7 sigma fragment expression |
| 59955 | pTHSSd_24 | PTac T3 sigma fragment expression |
| 59956 | pTHSSd_25 | PTac K1FR sigma fragment expression |
| 59957 | pTHSSd_26 | PTac CGG sigma fragment expression |
| 59958 | pTHSSd_27 | PTac null fragment (sigma CGG Y639A) expression |
| 59959 | pTHSSd_29 | PTac alpha fragment expression |
| 59960 | pTHSSd_34 | PTac GFP expression |
| 59961 | pTHSSd_38 | High core fragment expression (high resource allocator) |
| 59962 | pTHSSd_39 | Low core fragment expression (low resource allocator) |
| 59963 | pTHSSd_40 | High core fragment expression without SynZIP |
| 59964 | pTHSSd_41 | High full length T7 RNAP* expression |
| 59965 | pTHSSd_42 | beta core fragment expression |
| 59966 | pTHSSd_44 | constitutive expression of T7 sigma fragment |