Integrating gene synthesis and microfluidic protein analysis for rapid protein engineering

The capability to rapidly design proteins with novel functions will have a significant impact on medicine, biotechnology and synthetic biology. Synthetic genes are becoming a commodity, but integrated approaches have yet to be developed that take full advantage of gene synthesis. We developed a solid-phase gene synthesis method based on asymmetric primer extension (APE) and coupled this process directly to high-throughput, on-chip protein expression, purification and characterization (via mechanically induced trapping of molecular interactions, MITOMI). By completely circumventing molecular cloning and cell-based steps, APE-MITOMI reduces the time between protein design and quantitative characterization to 3-4 days. With APE-MITOMI we synthesized and characterized over 400 zinc-finger (ZF) transcription factors (TF), showing that although ZF TFs can be readily engineered to recognize a particular DNA sequence, engineering the precise binding energy landscape remains challenging. We also found that it is possible to engineer ZF-DNA affinity precisely and independently of sequence specificity and that in silico modeling can explain some of the observed affinity differences. APE-MITOMI is a generic approach that should facilitate fundamental studies in protein biophysics, and protein design/engineering.

Integrating Gene Synthesis and MITOMI for Rapid Protein Engineering

Matthew Christopher Blackburn

The research described within this thesis is primarily motivated by two fields of science with reversed, yet complementary, approaches to addressing the same essential objective: manipulating and understanding the complex program of life. Whereas synthetic biology has to do with the bottom-up construction of living systems from a library of âpartsâ, systems biology takes a top-down, reverse-engineering analysis of the same processes within functioning cells to elucidate the crucial elements and interactions. Both perspectives have led to important contributions in learning how biological systems operate. Discoveries from these studies can have far-reaching implications, notably in medicine, manufacturing, energy, and the environment. One exciting outcome of the research efforts within synthetic biology is the ability to design genetic constructs encoding proteins with novel, optimized functions. Although advances have been made in predicting how a proteinâs amino acid sequence relates to its higher order structural form and behavior, experimental methods for protein engineering remain invaluable for evaluating computationally derived designs. Unfortunately, the design-build-test cycle for rational protein development is commonly a time, labor and resource intensive endeavor. This is primarily due to limitations in the ability to generate libraries of gene variants and bottlenecks in cell-based expression and quantitative screening of protein products. This thesis describes the development of a solid-phase gene assembly technique and its integration with microfluidic protein analysis to accelerate the prototyping of novel protein designs. The gene assembly method utilizes commodity-scale, chemically manufactured DNA, and operates without the need for ligase or restriction enzymes. As a bench top process amenable to scale up, the modularity of the assembly process allows the efficient and economical generation of expression-ready gene variants. We directly coupled this gene assembly pipeline to a microfluidic device to enable high-throughput, on-chip, cell-free protein expression, purification, and characterization. By circumventing molecular cloning and cell-based steps, the lag time between protein design and quantitative analysis was dramatically reduced. As a proof of concept, this protein engineering platform was applied towards the construction of over 400 artificial, engineered variants of C2H2 zinc finger (ZF) proteins. ZF protein domains are the most prevalent form of transcription factor (TF) in humans, and are found throughout the tree of life. They provide a convenient structure for refactoring due to their relatively small size and composability, and are an ideal model for exploring the biophysics of TF-DNA specificity. The ability to engineer ZF proteins makes them useful as programmable, DNA-targeting units for applications in biotechnology and synthetic biology. We demonstrate that although ZFPs can be readily engineered to recognize a particular DNA target, engineering the precise binding energy landscape remains a challenge. Additionally, we show that ZF-DNA binding affinity can be tuned independently of sequence specificity. Together, these results demonstrate the versatility of the coupled gene assembly and microfluidic analysis platform as a new tool for rational protein engineering.

EPFL2016

Exploring on Protein-DNA interactions

Alexandra Styliani Kalantzi

A variety of DNA-binding proteins organizes the chromosomal DNA and regulates gene transcription, and DNA replication and recombination. In particular, for gene regulation there is a category of DNA-binding proteins, the transcription factors, which can detect and bind to a specific set of DNA motifs. For the protein-DNA complexes there are some solved structures, but for some cases, it is difficult to define experimentally these structures at the atomistic level. However, there are various in silico methods, that alone or in combination with experimental techniques, are capable of predicting proteins structures with high accuracy. DNA-binding proteins contain DNA-binding domains and unfortunately, there are few protein-DNA complexes that have been characterized at atomistic resolution. Here we have developed artificial neural networks to predict the binding sites and the interface between proteins and the DNA, based on a structure-based approach. Specifically, we used different interface descriptors and we included a variety of protein-DNA complexes, in order to predict the correct localizations of the DNA on a protein. A large group of proteins that can recognize specific DNA sequences is the KRAB-ZFP family. DNA recognition by zinc finger proteins (ZFPs) plays an important role in gene regulation. However, the molecular determinants defining the recognition of specific DNA nucleotides by ZFP finger repeats has not yet been decoded. Here, we present a method that can predict which ZF repeats specifically bind to a DNA target sequence and what is the most probable DNA target sequence for these repeats. Our method is based on the structural analysis of the binding network of resolved protein DNA complexes, and is validated on a benchmark set of solved ZFP-DNA complexes. We also characterized the binding specificity of two KRAB-ZFPs (ZFP14 and ZNF145) integrating our predictions with SMiLE-seq (Selective Microfluidics-based Ligand Enrichment followed by sequencing) data. Subsequently, we use our recognition code on ChIP-exo data, in order to give an insight into the poly-ZF domains binding patterns. We determined the most probable binding motifs and we separated the ZFPs in two groups: the ones that have a potential canonical and non-canonical binding. Altogether, this work gives an insight into the proteins that interact with the DNA, through a better understanding of the various protein-DNA interfaces and the predictions of these complexes at the atomistic level.

EPFL2019

Integrated microfluidic tools for the characterization of protein/DNA interactions in vitro and in vivo

Riccardo Dainese

The specific interaction between DNA and proteins constitutes one of the crucial elements in the regulation of gene expression. This thesis focuses on the development and optimization of two microfluidic-based technologies called SMiLE-seq and FloChIP that enable the sensitive and high-throughput analysis of two important aspects of protein/DNA interactions: respectively, 1) the transcription factor DNA-binding specificities and 2) the genome-wide distribution of chromatin-associated proteins. In Chapter 1, we describe the core motivation, operating principles, optimization and results obtained with SMiLE-seq. As opposed to existing solutions, SMiLE-seq offers the possibility to screen a large library of randomized DNA for sequence-specific protein ligands in a miniaturized context. SMiLE-seq integrates in a single microfluidic chip the advantages of both HT-SELEX and MITOMI. We first demonstrate as proof-of-principle that SMiLE-seq successfully recapitulates with robustness and reproducibility the binding specificities of known factors belonging to different species and TF families. Subsequently, we target the numerous although largely uncharacterized family of TFs called KRAB-ZFPs. We initially set out to redesign the microfluidic and protocol architecture in order to attain maximal throughput and sensitivity. Next, we add the ability of probing the sensitivity of these factors to methylation by synthesizing randomized methylated DNA libraries. Finally, we proceed to test 101 KRAB-ZFPs with both methylated and non-methylated DNA. We obtain high confidence motifs for 43 factors, of which 22 we not sensitive to methylation, 10 yielded motifs only with methylated DNA and 10 only with non-methylated DNA. By integrating our SMiLE-seq data with published ChIP-exo data and in silico predictions, we develop a framework for systematically identifying which zinc fingers directly contribute to DNA binding for a given KRAB-ZFPs. In Chapter 2, we describe the development and results obtained with FloChIP, a microfluidic implementation of the widespread ChIP-seq and sequential ChIP-seq protocols. FloChIP encompasses three main technological advances: 1) the multi-layered serface chemistry that allows any off-the-shelf antibody to be immobilized on-chip and and 2) a micropillar architecture that provides high surface-to-volume ratio and efficient removal of non-specific DNA. 3) The direct on-chip tagmentation of immunoprecipitated DNA. We validate our approach by first performing H3k27ac ChIP-seq on different number of sample inputs, i.e. from 500 to 10^6 cells. Next, we proceed to prove the flexibility of our approach by performing ChIP-seq on different histone marks â namely H3k27ac, H3k27me3, H3k4me3, H3k4me1 and H3k9me3 â and comparing them to existing ENCODE data. Both region-specific profiles and enrichment scores show high similarity with publicly available data. Subsequently, we set out to demonstrate the feasibility of on-chip sequential-ChIP-seq by performing consecutive H3k4me3 and H3k27me3 IP on chromatin derived from mouse embryonic stem cells. Finally, we illustrate the high-throughput feature of our technology by performing MEF2A-ChIP-seq on chromatin derived from 32 different lymphoblastoid cell lines.

EPFL2019

Integrating Gene Synthesis and MITOMI for Rapid Protein Engineering

Matthew Christopher Blackburn

EPFL2016

Exploring on Protein-DNA interactions

Alexandra Styliani Kalantzi

EPFL2019

Integrated microfluidic tools for the characterization of protein/DNA interactions in vitro and in vivo

Riccardo Dainese

EPFL2019