bioRxiv Science⌕ Search

Biology subjects

Henrike Niederholtmeyer

Publications and source records attributed to Henrike Niederholtmeyer.

2 recordsLinked to original sources
bioRxiv2015

GreA and GreB enhance Escherichia coli RNA polymerase transcription rate in a reconstituted transcription-translation system

Cell-free environments are becoming viable alternatives for implementing biological networks in synthetic biology. The reconstituted cell-free expression system (PURE) allows characterization of genetic networks under defined conditions but its applicability to native bacterial promoters and endogenous genetic networks is limited due to the poor transcription rate of Escherichia coli RNA polymerase in this minimal system. We found that addition of transcription elongation factors GreA and GreB to the PURE system increased transcription rates of E. coli RNA polymerase from sigma factor 70 promoters up to 6-fold and enhanced the performance of a genetic network. Furthermore, we reconstituted activation of natural E. coli promoters controlling flagella biosynthesis by the transcriptional activator FlhDC and sigma factor 28. Addition of GreA/GreB to the PURE system allows efficient expression from natural and synthetic E. coli promoters and characterization of their regulation in minimal and defined reaction conditions making the PURE system more broadly applicable to study genetic networks and bottom-up synthetic biology.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC=\"FIGDIR/small/024604_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (13K):\norg.highwire.dtl.DTLVardef@53902org.highwire.dtl.DTLVardef@e1ba85org.highwire.dtl.DTLVardef@19c961eorg.highwire.dtl.DTLVardef@143928d_HPS_FORMAT_FIGEXP M_FIG Graphical abstract\n\nC_FIG

Synthetic Biology↗
bioRxiv2015

A cell-free framework for biological systems engineering

SUMMARYWhile complex dynamic biological networks control gene expression and metabolism in all living organisms, engineering comparable synthetic networks remains challenging1,2. Conducting extensive, quantitative and rapid characterization during the design and implementation process of synthetic networks is currently severely limited due to cumbersome molecular cloning and the difficulties associated with measuring parts, components and systems in cellular hosts. Engineering gene networks in a cell-free environment promises to be an efficient and effective approach to rapidly develop novel biological systems and understand their operating regimes3-5. However, it remains questionable whether complex synthetic networks behave similarly in cells and a cell-free environment, which is critical for in vitro approaches to be of significance to biological engineering. Here we show that synthetic dynamic networks can be readily implemented, characterized, and engineered in a cell-free framework and consequently transferred to cellular hosts. We implemented and characterized the \"repressilator\"6, a three-node negative feedback oscillator in vitro. We then used our cell-free framework to engineer novel three-node, four-node, and five-node negative feedback architectures going from the characterization of circuit components to the rapid analysis of complete networks. We validated our cell-free approach by transferring these novel three-node and five-node oscillators to Escherichia coli, resulting in robust and synchronized oscillations reflecting the in vitro observation. We demonstrate that comprehensive circuit engineering can be performed in a cell-free system and that the in vitro results have direct applicability in vivo. Cell-free synthetic biology thus has the potential to drastically speed up design-build-test cycles in biological engineering and enable the quantitative characterization of synthetic and natural networks.

Synthetic Biology↗