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.

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