Lukmaan A. Bawazer

Measurements of translation initiation from all 64 codons in E. coli

Abstract Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the ‘start codons’ for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.

Efficient selection of biomineralizing DNA aptamers using deep sequencing and population clustering.

DNA-based information systems drive the combinatorial optimization processes of natural evolution, including the evolution of biominerals. Advances in high-throughput DNA sequencing expand the power of DNA as a potential information platform for combinatorial engineering, but many applications remain to be developed due in part to the challenge of handling large amounts of sequence data. Here we employ high-throughput sequencing and a recently developed clustering method (AutoSOME) to identify single-stranded DNA sequence families that bind specifically to ZnO semiconductor mineral surfaces. These sequences were enriched from a diverse DNA library after a single round of screening, whereas previous screening approaches typically require 5-15 rounds of enrichment for effective sequence identification. The consensus sequence of the largest cluster was poly d(T)30. This consensus sequence exhibited clear aptamer behavior and was shown to promote the synthesis of crystalline ZnO from aqueous solution at near-neutral pH. This activity is significant, as the crystalline form of this wide-bandgap semiconductor is not typically amenable to solution synthesis in this pH range. High-resolution TEM revealed that this DNA synthesis route yields ZnO nanoparticles with an amorphous-crystalline core-shell structure, suggesting that the mechanism of mineralization involves nanoscale coacervation around the DNA template. We thus demonstrate that our new method, termed Single round Enrichment of Ligands by deep Sequencing (SEL-Seq), can facilitate biomimetic synthesis of technological nanomaterials by accelerating combinatorial selection of biomolecular-mineral interactions. Moreover, by enabling direct characterization of sequence family demographics, we anticipate that SEL-Seq will enhance aptamer discovery in applications employing additional rounds of screening.

Combinatorial microfluidic droplet engineering for biomimetic material synthesis

Combinatorial chemical evolution is used to select oil-water droplet interfaces that drive inorganic nanoparticle synthesis. Although droplet-based systems are used in a wide range of technologies, opportunities for systematically customizing their interface chemistries remain relatively unexplored. This article describes a new microfluidic strategy for rapidly tailoring emulsion droplet compositions and properties. The approach uses a simple platform for screening arrays of droplet-based microfluidic devices and couples this with combinatorial selection of the droplet compositions. Through the application of genetic algorithms over multiple screening rounds, droplets with target properties can be rapidly generated. The potential of this method is demonstrated by creating droplets with enhanced stability, where this is achieved by selecting carrier fluid chemistries that promote titanium dioxide formation at the droplet interfaces. The interface is a mixture of amorphous and crystalline phases, and the resulting composite droplets are biocompatible, supporting in vitro protein expression in their interiors. This general strategy will find widespread application in advancing emulsion properties for use in chemistry, biology, materials, and medicine.

Genetic Algorithm‐Guided Discovery of Additive Combinations That Direct Quantum Dot Assembly

The use of combinations of organic additives to control crystallization, as occurs in biomineralization, is rarely investigated due to the vast potential reaction space. It is demonstrated here that combinatorial approaches led by genetic algorithm heuristics can enable identification of active additive combinations, and four key organic molecules are rapidly identified, which generate highly fluorescent CdS quantum dot superstructures.

Rapid preparation of highly reliable PDMS double emulsion microfluidic devices

This article presents a simple and highly reliable method for preparing PDMS microfluidic double emulsion devices that employs a single-step oxidative plasma treatment to both pattern the wettability of the microchannels and to bond the chips. As a key component of our strategy we use epoxy glue to define the required hydrophobic zones and then remove this after plasma treatment, but prior to bonding. This novel approach achieves surface modification and device sealing in a single process, which reduces chip preparation times to minutes and eliminates the need for unreliable coating processes. The second key element of our procedure is the maintenance of the patterned surfaces, where we demonstrate that immediate filling of the prepared device with water or the use of solvent-extracted PDMS vastly extends the operational lifetimes of the chips. The reliability of this technique is confirmed by generating water-in-oil-in-water (W/O/W) double emulsion droplets with controlled core/shell structures and volumes, and diameters as small as 55 μm. Its versatility is shown by simply using a different placement of the epoxy glue on the same chip design to create oil-in-water-in-oil (O/W/O) double emulsion droplets. Both W/O/W and O/W/O double emulsion droplets can therefore be created from the same soft-lithography mould. This simple method overcomes one of the key problems limiting the wider use of double emulsions – lack of reliability – while its speed and simplicity will facilitate the high-throughput production of monodisperse double emulsions. It could also be readily extended to produce microfluidic chips with more complex hydrophilic and hydrophobic patterns.

Enzymatically-controlled biomimetic synthesis of titania/protein hybrid thin films

Although it is widely recognised that enzymes play a significant role in sculpting complex silica skeletons in marine sponges, the potential for exploiting enzymes in materials synthesis has not yet been fully harnessed. In this work we show that the digestive enzyme papain can self-assemble into monolayers on oxide surfaces, where they then drive the formation of crystalline titanium dioxide nanoparticles. This dual functionality of thin film formation and mineralization promotion has the potential to enable the construction of hierarchical inorganic/organic structures in the form of continuous amorphous titania/protein films which can be refined to 93% anatase post annealing. Additional control over the film thickness is afforded by layer-by-layer processing using a simple dip-coating approach. Papains TiO2-mineralizing activity displays complex kinetics that deviates from the native Michaelis-Menten kinetic activity, yet deactivation studies demonstrate that this activity relies upon residues that are essential for catalytic site function. These parameters provide unique insight into enzymatic biomineralization, allowing a flexible route to achieving bioengineered titania heterostructures, and potentially providing a green-chemistry solution to photovoltaic cell development.