Abstract
Expanding the genetic toolbox for prokaryotic synthetic biology is a promising strategy for enhancing the dynamic range of gene expression and enabling new engineered applications for research and biomedicine. Here, we reverse the current trend of moving genetic parts from prokaryotes to eukaryotes and demonstrate that the activating eukaryotic transcription factor QF and its corresponding DNA-binding sequence can be moved to E. coli to introduce transcriptional activation, in addition to tight off states. We further demonstrate that the QF transcription factor can be used in genetic devices that respond to low input levels with robust and sustained output signals. Collectively, we show that eukaryotic gene regulator elements are functional in prokaryotes and establish a versatile and broadly applicable approach for constructing genetic circuits with complex functions. These genetic tools hold the potential to improve biotechnology applications for medical science and research.
Highlights
Expanding the genetic toolbox for prokaryotic synthetic biology is a promising strategy for enhancing the dynamic range of gene expression and enabling new engineered applications for research and biomedicine
When T7 RNA polymerase (T7RNAP) is present upon isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, the eukaryotic transcription factor QF can activate gene expression in bacteria beyond baseline levels
Developing unique genetic circuits and repurposing genetic regulatory elements to reprogram cellular function has led to new opportunities for clinical applications[48,49] and has the potential to influence the therapeutic response to public health crises
Summary
Expanding the genetic toolbox for prokaryotic synthetic biology is a promising strategy for enhancing the dynamic range of gene expression and enabling new engineered applications for research and biomedicine. We show that eukaryotic gene regulator elements are functional in prokaryotes and establish a versatile and broadly applicable approach for constructing genetic circuits with complex functions. Transcription factors are DNA-binding proteins that repress or activate transcription by binding to a specific DNA sequence within the genome, and they have been widely used to build genetic circuits that regulate gene expression. Because T7RNAP is highly selective for the T7 promoter, transcription of downstream genes is selective[25] This system can be made inducible by controlling the transcription of T7RNAP using bacterial systems such as AraC26, LacI27, and TetR14 and placing their respective DNA-binding sites upstream of an endogenous promoter that drives the expression of T7RNAP. We reversed the conventional approach of moving gene regulatory parts from prokaryotes to eukaryotes by moving the activating eukaryotic transcription factor QF and its corresponding DNA-binding site, QUAS, from the filamentous fungus Neurospora crassa to bacteria. A second generation of the QF transcription factor (QF2) that has the middle region of the protein removed to decrease toxicity in other organisms was used in this study[31]
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