Single Molecule Conformational Memory Extraction: P5ab RNA Hairpin

Extracting kinetic models from single molecule data is an important route to mechanistic insight in biophysics, chemistry, and biology. Data collected from force spectroscopy can probe discrete hops of a single molecule between different conformational states. Model extraction from such data is a challenging inverse problem because single molecule data are noisy and rich in structure. Standard modeling methods normally assume (i) a prespecified number of discrete states and (ii) that transitions between states are Markovian. The data set is then fit to this predetermined model to find a handful of rates describing the transitions between states. We show that it is unnecessary to assume either (i) or (ii) and focus our analysis on the zipping/unzipping transitions of an RNA hairpin. The key is in starting with a very broad class of non-Markov models in order to let the data guide us toward the best model from this very broad class. Our method suggests that there exists a folding intermediate for the P5ab RNA hairpin whose zipping/unzipping is monitored by force spectroscopy experiments. This intermediate would not have been resolved if a Markov model had been assumed from the onset. We compare the merits of our method with those of others

A Method for Multiplex Gene Synthesis Employing Error Correction Based on Expression

<div><p>Our ability to engineer organisms with new biosynthetic pathways and genetic circuits is limited by the availability of protein characterization data and the cost of synthetic DNA. With new tools for reading and writing DNA, there are opportunities for scalable assays that more efficiently and cost effectively mine for biochemical protein characteristics. To that end, we have developed the Multiplex Library Synthesis and Expression Correction (MuLSEC) method for rapid assembly, error correction, and expression characterization of many genes as a pooled library. This methodology enables gene synthesis from microarray-synthesized oligonucleotide pools with a one-pot technique, eliminating the need for robotic liquid handling. Post assembly, the gene library is subjected to an ampicillin based quality control selection, which serves as both an error correction step and a selection for proteins that are properly expressed and folded in <i>E</i>. <i>coli</i>. Next generation sequencing of post selection DNA enables quantitative analysis of gene expression characteristics. We demonstrate the feasibility of this approach by building and testing over 90 genes for empirical evidence of soluble expression. This technique reduces the problem of part characterization to multiplex oligonucleotide synthesis and deep sequencing, two technologies under extensive development with projected cost reduction.</p></div

Multiplex Library Synthesis of Genes.

<p>(A) Individual genes (top) are assembled from a set of oligonucleotides (arrows) with 15 bp of unique overlapping sequence. Synthons include universal forward (UF) and reverse (UR) primer binding regions common for all genes in the library, and specific forward (SF) and reverse (SR) sites for amplification of individual genes. Synthons include restriction endonuclease sites (RE1 = EcoRI, RE2 = BamHI) for downstream cloning purposes, a random filler sequence (Fi) for length standardization, and are < 950 bp long. (B) Gene synthesis from a pooled library of oligonucleotide starts with high temperature annealing and ligation, then individual genes can either be amplified with SF/SR primers (left) or library of genes is synthesized and amplified (right) with emulsion PCR using UF/UR primers with annealing 5’ and 3’ overhangs to enable suppression PCR, which preferentially amplifies longer synthons. (C) Excitation of 87 GFP variants successfully synthesized, amplified individually, then expressed in <i>E</i>. <i>coli</i> show functional fluorescent protein at various emission wavelengths. Images are combined from ultraviolet (top layer, 50% transparency) and blue light illumination (470nm) with no emission filtering. White X represents the negative control well with media only.</p

Expression correction for properly folded soluble proteins.

<p>(A) The synthesized gene library open reading frames (ORFs) are cloned via RE1/RE2 into a vector containing an N-terminal Tat pathway secretion signal (ssTorA) and a C-terminal TEM-1 β-lactamase. (B) Synthesized genes that are expressed, properly folded, and soluble result in the export of mature β-lactamase fusions to the <i>E</i>. <i>coli</i> periplasm, which confers ampicillin resistance. Selection against insoluble protein serves to both remove correct gene sequences which are poorly expressed in <i>E</i>. <i>coli</i> and remove incorrect gene synthesis products as the resulting protein is often insoluble. Expression profiling is characterized with Illumina MiSeq next generation sequencing.</p

Individual Gene Performance in Expression Correction Assay on Ampicillin.

<p>(A) Fold killing on 100 μg/mL of ampicillin (left axis, gray bars) and fraction of soluble protein (right axis, red line) for each individual gene shows an increase in cell survival with increasing soluble protein fraction. Fold killing is calculated based on viable colony counts on solid media with no ampicillin divided by counts at 100 μg/mL ampicillin. Soluble protein fraction was quantified from western blotting of soluble and insoluble protein fractions expressed as C-terminal FLAG tag fusions. (n = 3 for both experiments). Error bars represent standard deviation. (B) Percent representation of each gene per ampicillin selection pool shows enrichment of genes which survive at 100 μg/mL of ampicillin and have higher soluble protein fractions in (A). Percent is calculated as the median number of reads per base pair of each gene compared to the sum of medians for each gene per ampicillin pool.</p