Rapid, Simple, and High-Throughput Antimicrobial Susceptibility Testing and Antibiotics Screening

In this paper is presented a system for determining density of bacteria and fungi in order to determine susceptibility to antibiotics. It is faster than traditional methods, requiring only 4-5 hours as opposed to 24-48 hours. The basic concept is that the surface of the bacteria, which is negatively charged, breaks apart an interaction of negatively charged fluorescein (FL) with positively charged polyfluorene (PFP). Breaking apart this interaction disrupts FRET between FL and PFP, and a change in emission is seen.

Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen

The authors of this paper have engineered a novel biological system that enables E.coli to sense and kill pathogenic P. aeruginosa. Given the growing resistance to current antibiotics, this study is an excellent example of a novel synthetic biology-driven antimicrobial strategy. It has the potential to be transferred to bacteria species of the human microbiota, arming them to fight pathogens on our behalf.

Amplification, Mutation, and Sequencing of a Six-Letter Synthetic Genetic System

In this paper the Benner group is able to show that they successfully added two nucleotides to artificial DNA.  Furthermore they present that they are able to perform PCR on the 6 nucleotide DNA incorporating the new bases.  Beyond that they can mutate the artificial DNA making it viable for evolution.  The expanded code could lead to several developments down the road including a way of incorporating unnatural amino acids into proteins.  Though there is still a lot of work before these artificial DNAs could be functional in a cell, but these findings increase the the possible number of codons from 64 to 216.

Diazo Reagents with Small Steric Footprints for Simultaneous Arming/ SAR Studies of Alcohol-Containing Natural Products via O-H Insertion

This paper describes the discovery of a new linker that facilitates target identification experiments. They synthesized a diazo reagent that is substituted in the alpha position with different electron-withdrawing groups and also with an alkyne. The diazo reagent can be attached to a natural product containing hydroxyl groups in an efficient and chemoselective way using Rhodium catalyst, this natural product can be subsequently tagged with biotin via click chemistry to facilitate affinity chromatography.
Most of the time, these experiments require a lot of structure activity relationship to find a position on the molecule where the linker can be attached without modifying the bioactivity. The really interesting thing about this paper is that the diazo reagent was selectively attached to the most accessible hydroxyl group of diverse natural products and the activity was maintained, yielding probes ready to be used in affinity chromatography without the need of SAR.

Designing a New Diels Alderase: A Combinatorial, Semirational Approach Including Dynamic Optimization

This article illuminates a combinatorial approach to the design of enzymes that catalyze Diels-Alder reactions. We have seen from Siegel et al. that computational design of the Diels-Alder reaction is viable. This paper seeks to improve the catalytic efficiency of the Diels-Alder design, DA_20_00 Siegel et al., through molecular modeling, molecular docking, and density functional theory. The computational screening method used optimizes the substrate and enzyme simulataneously. In contrast to the Siegel et al. approach, a predesigned active sight is not incorporated into an existing protein fold; limiting mutations. These two approaches provide greater insight into de novo Diels-Alder design.

Directed Evolution of the Nonribosomal Peptide Synthetase AdmK Generates New Andrimid Derivatives In Vivo

Many lead compounds in the search for new drugs derive from peptides and polyketides whose similar biosynthetic enzymes have been difficult to engineer for production of new derivatives. Problems with generating multiple analogs in a single experiment along with lack of high-throughput methods for structure-based screening have slowed progress inthis area.

However, by using directed evolution and a multiplexed assay to screen a library of >14,000 members, it is able to generate three derivatives of the antibacterial compound, andrimid. Since another limiting factor in reengineering these mega-enzymes of secondary metabolism has been that commonly used hosts such as Escherichia coli often give lower product titers. However, in this study, reengineering was performed in the native producer, Pantoea agglomerans. This integrated in vivo approach can be extended to larger enzymes to create analogs of natural products for bioactivity testing.

A natural prodrug activation mechanism in nonribosomal peptide synthesis

A new mechanism for the cleavage and activation of nonribosomally made peptides and peptide-polyketide hybrids that are apparently operational in several different bacteria has been identified. This process includes the cleavage of a precursor molecule by a membrane-bound and D-asparagine–specific peptidase, as shown here in the biosynthesis of the antibiotic xenocoumacin from Xenorhabdus nematophila.

A De Novo Protein Binding Pair By Computational Design and Directed Evolution

This article presents an effective combination of computational design and directed evolution to synthesize a de novo protein binding pair. Karanicolas and coworkers first selected two proteins with complementary surface features but no natural affinity to bind one another, then using a 'motif based' strategy were able to model new favorable binding interactions. This method differs from the standard technique of importing known binding elements from natural protein binding pairs. The computational models can then be synthesized and enhanced. Using directed evolution, the binding affinity of the protein complex was increased, yielding a KD of 180 pM. This result is better than any previous computer-designed protein binding pair and highlights the strength of combining computational and directed evolution techniques.

Anthracene-BODIPY Dyads as Fluorescent Sensors for Biocatalytic Diels-Alder Reactions

In Gavin's class Rob gave a presentation on de novo computational design of a Diels-Alderase. The enzyme produced in this research had relatively low activity when compared to normal enzymes. Improving the activity of this de novo enzyme is limited by the development of screen/selection.
This paper describes a fluorescent sensor for a monitoring a ribozyme Diels-Alderase. They use anthracene attached to a BODIPY fluorophore. This sensor could be a good tool (or a starting point) for improving the activity of other alderases including the de novo enzyme developed by Siegel et al. (Science. 2010 Jul 16;329(5989):309-13).

Using Modularly Assembled Ligands To Bind RNA Internal Loops Separated by Different Distances

This paper is related to fragment-based drug discovery in that fragments which are effective at binding the target can be connected by a linker of appropriate length to increase affinity and specificity. The goal of this research was to determine which combinations of linker length and distance between base pairs of hairpin RNA yielded the highest affinity and selectivity. This strategy may best be applied to RNA targets, but the general principles could be extended to protein targets.