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A 'resource allocator' for transcription based on a highly fragmented T7 RNA polymerase.
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.

Voigt Lab Fragmented T7 RNAP Plasmids

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
59947pTHSSd_1Splitposon in KanR ColE1 backbone
59948pTHSSd_8PT7 GFP reporter
59949pTHSSd_9PT3 GFP reporter
59950pTHSSd_10PK1F GFP reporter
59951pTHSSd_11PCGG GFP reporter
59952pTHSSd_14Triple split (at 67, 601-SZ)
59953pTHSSd_18Quad split (at 61, 179, 601-SZ)
59954pTHSSd_23PTac T7 sigma fragment expression
59955pTHSSd_24PTac T3 sigma fragment expression
59956pTHSSd_25PTac K1FR sigma fragment expression
59957pTHSSd_26PTac CGG sigma fragment expression
59958pTHSSd_27PTac null fragment (sigma CGG Y639A) expression
59959pTHSSd_29PTac alpha fragment expression
59960pTHSSd_34PTac GFP expression
59961pTHSSd_38High core fragment expression (high resource allocator)
59962pTHSSd_39Low core fragment expression (low resource allocator)
59963pTHSSd_40High core fragment expression without SynZIP
59964pTHSSd_41High full length T7 RNAP* expression
59965pTHSSd_42beta core fragment expression
59966pTHSSd_44constitutive expression of T7 sigma fragment

Antibodies from Article