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bioRxiv · 10.1101/2025.10.08.680606

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

Kemp, P.·Blat Belmonte, M.·Schmidt, M.·Eisenhauer, K.·Gebhardt, C.·Kabuth, K.·Koeppl, H.·Stein, V.
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Abstract

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.

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Kemp, P., Blat Belmonte, M., Schmidt, M., Eisenhauer, K., Gebhardt, C., Kabuth, K., Koeppl, H., Stein, V.. 2025-10-08. iFLinkC-EZ: A scalable and automatable method for the assembly of complex fusion proteins and multi-gene expression constructs based on the iFLinkC framework. https://doi.org/10.1101/2025.10.08.680606

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