Mutation Maker, An Open Source Oligo Design Platform for Protein Engineering.

Protein engineering is the discipline of developing useful proteins for applications in research, therapeutic, and industrial processes by modification of naturally occurring proteins or by invention of de novo proteins. Modern protein engineering relies on the ability to rapidly generate and screen diverse libraries of mutant proteins. However, design of mutant libraries is typically hampered by scale and complexity, necessitating development of advanced automation and optimization tools that can improve efficiency and accuracy. At present, automated library design tools are functionally limited or not freely available. To address these issues, we developed Mutation Maker, an open source mutagenic oligo design software for large-scale protein engineering experiments. Mutation Maker is not only specifically tailored to multisite random and directed mutagenesis protocols, but also pioneers bespoke mutagenic oligo design for de novo gene synthesis workflows. Enabled by a novel bundle of orchestrated heuristics, optimization, constraint-satisfaction and backtracking algorithms, Mutation Maker offers a versatile toolbox for gene diversification design at industrial scale. Supported by in silico simulations and compelling experimental validation data, Mutation Maker oligos produce diverse gene libraries at high success rates irrespective of genes or vectors used. Finally, Mutation Maker was created as an extensible platform on the notion that directed evolution techniques will continue to evolve and revolutionize current and future-oriented applications.

The thymidylate kinase genes from Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus confer 3'-azido-3'-deoxythymidine resistance to Escherichia coli.

The case number of invasive multidrug-resistant bacteria cultured from both hospital and community acquired infections is increasing at an alarming rate. Identifying the mechanisms bacteria use to escape the current antimicrobial treatments is essential to containing potential outbreaks and developing new antimicrobial therapies. Many bacteria naturally encode nonessential resistance genes on their chromosome enabling their survival and/or persistence in the presence of antibiotics using enzymes and efflux pumps. This study investigates the ability of an evolutionarily conserved essential gene to provide resistance against antimicrobial compounds. An Escherichia coli chromosomally encoded thymidylate kinase (tmk) conditional lethal strain was developed to investigate tmk alleles from relevant nosocomial pathogens. The thymidylate kinase conditional lethal strain harboring a plasmid with a tmk gene from Mycobacterium tuberculosis, methicillin-resistant Staphylococcus aureus (MRSA), or Pseudomonas aeruginosa downstream of an inducible promoter was examined for survival against increasing concentrations of 3'-azido-3'-deoxythymidine (AZT). The results indicate that M. tuberculosis and MRSA thymidylate kinases are deficient in cellular activity toward AZT monophosphate.

RNA visualization in bacteria by fluorescence in situ hybridization.

Detecting localized RNA in bacteria is difficult due to the properties of RNA and the small size of the cell. Fluorescence in situ hybridization (FISH) has been an invaluable method for detecting and imaging RNA. In FISH, RNA is fixed in its native subcellular position through chemical cross-linking. An oligonucleotide probe conjugated to a fluorophore is annealed to the target RNA, and the target RNA/probe hybrid is visualized using fluorescence microscopy. This chapter describes the use of FISH to visualize tmRNA, a regulatory RNA required for trans-translation. The method can be adapted to visualize the localization of other regulatory and messenger RNAs as well.

Subcellular localization of a bacterial regulatory RNA.

Eukaryotes and bacteria regulate the activity of some proteins by localizing them to discrete subcellular structures, and eukaryotes localize some RNAs for the same purpose. To explore whether bacteria also spatially regulate RNAs, the localization of tmRNA was determined using fluorescence in situ hybridization. tmRNA is a small regulatory RNA that is ubiquitous in bacteria and that interacts with translating ribosomes in a reaction known as trans-translation. In Caulobacter crescentus, tmRNA was localized in a cell-cycle-dependent manner. In G(1)-phase cells, tmRNA was found in regularly spaced foci indicative of a helix-like structure. After initiation of DNA replication, most of the tmRNA was degraded, and the remaining molecules were spread throughout the cytoplasm. Immunofluorescence assays showed that SmpB, a protein that binds tightly to tmRNA, was colocalized with tmRNA in the helix-like pattern. RNase R, the nuclease that degrades tmRNA, was localized in a helix-like pattern that was separate from the SmpB-tmRNA complex. These results suggest a model in which tmRNA-SmpB is localized to sequester tmRNA from RNase R, and localization might also regulate tmRNA-SmpB interactions with ribosomes.

Screen for localized proteins in Caulobacter crescentus.

Precise localization of individual proteins is required for processes such as motility, chemotaxis, cell-cycle progression, and cell division in bacteria, but the number of proteins that are localized in bacterial species is not known. A screen based on transposon mutagenesis and fluorescence activated cell sorting was devised to identify large numbers of localized proteins, and employed in Caulobacter crescentus. From a sample of the clones isolated in the screen, eleven proteins with no previously characterized localization in C. crescentus were identified, including six hypothetical proteins. The localized hypothetical proteins included one protein that was localized in a helix-like structure, and two proteins for which the localization changed as a function of the cell cycle, suggesting that complex three-dimensional patterns and cell cycle-dependent localization are likely to be common in bacteria. Other mutants produced localized fusion proteins even though the transposon has inserted near the 5' end of a gene, demonstrating that short peptides can contain sufficient information to localize bacterial proteins. The screen described here could be used in most bacterial species.

Peptide signals encode protein localization.

Many bacterial proteins are localized to precise intracellular locations, but in most cases the mechanism for encoding localization information is not known. Screening libraries of peptides fused to green fluorescent protein identified sequences that directed the protein to helical structures or to midcell. These peptides indicate that protein localization can be encoded in 20-amino-acid peptides instead of complex protein-protein interactions and raise the possibility that the location of a protein within the cell could be predicted from bioinformatic data.