Kemp, P. | bioRxiv Science

bioRxiv2025

iFLinkC-EZ: A scalable and automatable method for the assembly of complex fusion proteins and multi-gene expression constructs based on the iFLinkC framework

Standardized methods for the assembly of DNA constructs have become indispensable in synthetic biology, biotechnology and basic research. The majority of frameworks - most prominently based on Golden Gate - have been developed and optimized for the assembly of transcriptional units (TUs) and thereof composed multi-gene expression constructs. This is significantly enabled by the modular organization of functional elements underlying any given TU. In contrast, the assembly of protein coding sequences typically necessitates tailored approaches. Addressing this technological gap, iterative functional linker cloning (iFLinkC) was recently developed to provide a standardized framework for assembling protein coding sequences of arbitrary size and complexity from the ground up based on pairwise ligations of any two DNA fragments via a two-base overlap. Yet, a need for physically purifying DNA fragments imposes significant procedural complexity and operator skill. Overcoming these limitations, a new assembly algorithm, termed iFLinkC-EZ, is presented. Crucially, the DNA assembly products are now purified by genetic means which greatly simplifies the underlying assembly process and also renders it compatible with robotic automation. Further, the potency of iFLinkC-EZ is demonstrated in the assembly of mono- and poly-chromatic poly-fluorescent fusion proteins as well as poly-cistronic expression constructs while examining the effect of poly-PT and poly-GGS linkers as well as ribosome binding sites on the functional expression of poly-fluorescent fusion protein constructs. Further, the utility of poly-fluorescent proteins is demonstrated for the enhanced labeling of surface displayed recombinant binders in yeast display. Given its ease and efficiency, iFLinkC-EZ is anticipated to be applicable to the assembly of many different types of fusion proteins and thereof based multi-gene expression constructs including but not limited to synthetic protein switches, sensors and multi-enzyme complexes as well as thereof composed genetic circuits and metabolic pathways.

synthetic biology↗

bioRxiv2022

Systematic Evaluation of Self-Assembling Membrane Peptides to Form Nanopores in the context of the Functional Nanopore Screen

The functional analysis of protein nanopores is typically conducted in planar lipid bilayers or liposomes exploiting high-resolution but low-throughput electrical and optical read-outs. Yet, the reconstitution of protein nanopores in vitro still constitutes an empiric and low-throughput process. Addressing these limitations, nanopores can now be analyzed using the functional nanopore (FuN) screen exploiting genetically-encoded fluorescent protein sensors that resolve distinct nanopore-dependent Ca2+ in- and efflux patterns across the inner membrane of Escherichia coli. With a primary proof-of-concept established for the S2168 holin, and thereof based recombinant nanopore assemblies, the question arises to what extent alternative nanopores can be analyzed with the FuN screen and to what extent alternative fluorescent protein sensors can be adapted. Focussing on self-assembling membrane peptides, three sets of 13 different nanopores are assessed for their capacity to form nanopores in the context of the FuN screen. Nanopores tested comprise both natural and computationally-designed nanopores. Further, the FuN screen is extended to K+-specific fluorescent protein sensors to provide a complementary read-out to Ca2+. Finally, a comparison with high-resolution biophysical and electrophysiological studies in planar lipid bilayers provides an experimental benchmark for future studies.

synthetic biology↗