em530 channel (GFP) detected protein/TF, bound by anti-GFP background subtraction Integrating Gene Synthesis and Microﬂuidics for MC Blackburn, E Petrova, SJ Maerkl Institute of Bioengineering, School of Engineering, EPFL, Lausanne, Switzerland Literature 1) de Raad M, et al. A solid-phase platform for combinatorial and scarless multipart gene assembly. ACS Syn Bio 2013. 2) Lunqvist M, et al. Solid-phase cloning for hight- throughput assmebly of single and multiple DNA parts. NAR Vol 43(7), 2015. 3) Wolfe SA, et al. DNA Recognition by Cys2His2 zinc ﬁnger proteins. Annu. Rev. Biophys. Biomol. Struct., 1999. 4) Beerli RR, et al. Engineering polydactyl sinc-ﬁnger transcription factors. Nature Biotech, Vol 20, 2002. 5) Khalil AS, et al. A synthetic biology framework for programming eukaryotic transcription functions. Cell Vol 150, 2012. 6) Persikov AV, et al. Deep sequencing of large library selections allows computational discovery of diverse sets of zinc ﬁngers that bind common targets. NAR Vol 42 (3), 2014. 7) Persikov AV, et al. A systematic survey of the Cys2His2 zinc ﬁnger DNA-binding landscape. NAR, Vol 43(3), 2015. 8) Najafabadi H, et al. C2H2 zinc ﬁnger proteins greatly expand the human regulatory lexicon. Nature Biotech, 2015. 9) Fordyce PM, et al. De novo identiﬁcation and biophysical characterization of transcription-factor binding sites with microﬂuidic afﬁnity analysis. Nature Biotech Vol 28 (9), 2010. 10) Maerkl SJ, et al. A systems approach to measuring the binding energy landscapes of transcription factors. Science Vol 315, 2007. Introduction Projects ZF Modular Combinatorics ZF Speciﬁcity Engineering Methods Objective Engineering synthetic proteins with desired functions is difﬁcult. It can require multiple rounds of computational design, gene construction, expression and functional characterization to produce a new protein which consistently behaves in a novel, expected manner. Traditionally, these time and resource-consuming cycles involve gene synthesis via ligation or PCR-based assembly of DNA parts, cloning, and sequencing to ensure the gene coding for a given engineered protein is error-free1. This is followed by an assay to determine if the protein folds correctly and how effective the protein is at performing its intended function. A ligation/plasmid/cloning-free gene synthesis technique (detailed below) for rapidly constructing genes from synthetic DNA parts was coupled with a 1024- chamber MITOMI device for in vitro characterization of engineered zinc ﬁnger transcription factors. DNA parts were designed such that single ﬁnger modules could be interchanged with others, enabling the combinatorial construction of many different proteins from a small DNA pool. Engineering the binding speciﬁcity of individual zinc ﬁnger arrays through controlled amino acid substitutions was examined as a proof-of-concept for protein design. Solution Conceive of and build a platform for rapidly building genes from short, synthetic DNA oligomers (under 90 nt), examine the error rate of synthesis, and generate many ‘prototypes’ of proteins for a high-throughput functional screen. Apply this technique to several rounds of design > synthesis > expression > characterization to evolve a protein for a desired application. Zinc ﬁnger modules from zif268 and previously characterized5 arrays 37-12, 92-1 and 158-2 were used to generate novel zinc ﬁnger arrays. All ﬁngers in each position were interchanged and tested against a target library containing the expected consensus were each ZF behaving independently of the others in the same array. Zif268 (wt) N C ﬁnger 1 ﬁnger 2 ﬁnger 3 RSDELTR RSDHLTT RSDERKR C N RKREDSR TTLHDSR RTLEDSR 5'- aGCG TGG GCGt 6 5 4 3 2 1 -1 6 5 4 3 2 1 -1 6 5 4 3 2 1 -1 37-12 N C ﬁnger 1 ﬁnger 2 ﬁnger 3 RNFILQR DRANLRR RHDQLTR C N RTLQDHR RRLNARD RQLIFNR 5'- tGAG GAC GTGt 6 5 4 3 2 1 -1 6 5 4 3 2 1 -1 6 5 4 3 2 1 -1 158-2 N C ﬁnger 1 ﬁnger 2 ﬁnger 3 DKTKLRV VRHNLTR QSTSLQR C N RQLSTSQ RTLNHRV VRLKTKD 5'- tGTA GAT