Artificial Sigma Factors Based on Bisected T7 RNA Polymerase

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FIG. 1 shows a non-limiting embodiment of a system for testing aspects of the invention, including a generator plasmid that produces a conserved T7 fragment and determines total transcriptional units in the system. In some embodiments, the generator plasmid is present in very low copy number. FIG. 1 also demonstrates an allocator plasmid, which produces variable T7 fragments and targets transcriptional units to specific promoters. In some embodiments, the allocator plasmid is present in medium copy number. FIG. 1 also demonstrates a reporter/effector plasmid, which contains promoters that are targeted by T7 variants and a desired output. In some embodiments, the reporter/effector plasmid is present in low copy number.FIG. 2 shows a graph of sigma-like fragment competition. When two sigma-like fragments were expressed with a constant level of the core fragment, there was a clear tradeoff in their activities. The T7 sigma-like fragment was expressed at a constant level and the T3 sigma-like fragment was expressed at varying levels. As the level of the T3 sigma-like fragment increased, activity at a T3 promoter specifically increased (white circles), while T7 specific promoter activity decreased (grey diamonds). The activity at each of these promoters is shown as a percentage of their maximum activity measured with the same amount of core fragment. The sum of these two normalized activities (black triangles) is very close to 100% over the expression range tested, indicating that the entirety of the core fragment pool is being allocated to the two sigma-like fragments.FIG. 3 shows a non-limiting example of modeling for systems described herein and uses of such systems. In this model, A is a core fragment, while B1 and B2 are sigma-like fragments.
Categories
Inventors
Professor Christopher Voigt
Department of Biological Engineering, MIT
External Link (mit.edu)
Thomas Segall-Shapiro
Department of Biological Engineering, MIT
Managed By
Jon Gilbert
MIT Technology Licensing Officer
Patent Protection

Artificial sigma factors based on bisected t7 rna polymerase

US Patent Pending 2015-0368625
Publications
A ‘resource allocator’ for transcription based on a highly fragmented T7 RNA polymerase
Molecular Systems Biology , Vol. 10, 742 (2014)

Applications

This invention is a control element for a genetic circuit which allows regulation with tradeoffs and the calculation of the ratio of two input signals.

Problem Addressed

Synthetic biology relies on regulation of gene expression which is difficult for complex functions. This invention is a control element for genetic circuits for predictable programming of biology.

Technology

This technology is based on T7 RNA polymerase which initiates transcription with high specificity allowing for orthogonal regulation.  This polymerase, which is transferable between cell types, has a conserved core and a variable promoter recognition region which controls specificity. This invention is a recombinant T7 RNA polymerase consisting of two separately expressed protein fragments, which were created by bisecting the protein at a library of random sites to identify locations where function is retained. The inventors have created a system of one conserved fragment, and up to four variable ones,  in which the variable proteins can bind the conserved protein and make it functionally target a promoter DNA sequence and initiate transcription.  Therefore, this invention may be used to implement a type of trade-off logic where expression of one pathway decreases as another increases, or as a ratio calculator that allows the measurement of the ratio between two input signals and returns a single protein expression level as a result. 

Advantages

  • Conserved protein amount sets the maximum transcriptional output from the system
  • Variable proteins allocate transcriptional potential
  • T7 RNA polymerases are widely used; invention can be used with many existing systems and cell types