Experimental Protocols for Generating Focused Mutant Libraries and Screening for Thermostable Proteins.

Many proteins are rapidly deactivated when exposed to high or even ambient temperatures. This cannot only impede the study of a particular protein, but also is one of the major reasons why enzyme catalysis is still widely unable to compete with established chemical processes. Furthermore, differences in protein stability are a challenge in synthetic biology, when individual modules prove to be incompatible. The targeted stabilization of proteins can overcome these hurdles, and protein engineering techniques are more and more reliably supported by computational chemistry tools. Accordingly, algorithms to predict the differences in folding energy of a mutant compared to the wild-type, ΔΔGfold, are used in the highly successful FRESCO workflow. The resulting single mutant prediction library consists typically of a few hundred amino acid exchanges, and after combining the most successful hits we so far obtained stabilized mutants which exhibited increases in apparent melting temperature of 20-35°C and showed vastly increased half-lives, as well as resistance to cosolvents. Here, we report a detailed protocol to generate these mutant libraries experimentally, covering the entire workflow from primer design, through mutagenesis, protein production and screening, to mutation combination strategies. The individual parts of the method are furthermore applicable to many other scenarios besides protein stabilization, and these protocols are valuable for any project requiring individual or semi high-throughput site-directed mutagenesis, protein expression and purification, or generation of mutant combination libraries.

Identification of Response Elements on Promoters Using Site-Directed Mutagenesis and Chromatin Immunoprecipitation.

Proximal promoters are located upstream of the transcription start sites of genes, and they contain regulatory sequences on which bind different transcription factors for promoting colorectal cancer progression. Here we describe the comprehensive methodology used previously for the identification and functional characterization of MYC-responsive elements in the integrin α1 subunit (ITGA1) gene using a combination of in silico analysis, site-directed mutagenesis, and chromatin immunoprecipitation.

Strategies used for genetically modifying bacterial genome: site-directed mutagenesis, gene inactivation, and gene over-expression.

With the availability of the whole genome sequence of Escherichia coli or Corynebacterium glutamicum, strategies for directed DNA manipulation have developed rapidly. DNA manipulation plays an important role in understanding the function of genes and in constructing novel engineering bacteria according to requirement. DNA manipulation involves modifying the autologous genes and expressing the heterogenous genes. Two alternative approaches, using electroporation linear DNA or recombinant suicide plasmid, allow a wide variety of DNA manipulation. However, the over-expression of the desired gene is generally executed via plasmid-mediation. The current review summarizes the common strategies used for genetically modifying E. coli and C. glutamicum genomes, and discusses the technical problem of multi-layered DNA manipulation. Strategies for gene over-expression via integrating into genome are proposed. This review is intended to be an accessible introduction to DNA manipulation within the bacterial genome for novices and a source of the latest experimental information for experienced investigators.

Gene knockouts, in vivo site-directed mutagenesis and other modifications using the delitto perfetto system in Saccharomyces cerevisiae.

Gene manipulation serves the purpose of providing a better understanding of the function of specific genes as well as for developing novel variants of the genes of interest. The generation of knockout genes, the alteration, depletion, or enhancement of a particular gene function through the generation of specific gene mutations, or the generation of random mutations in a gene are all essential processes for gene manipulation. The genome of the yeast Saccharomyces cerevisiae is relatively easy to modify, owing to its efficient homologous recombination (HR) system. Gene knockout can be a very simple, one-step approach to eliminate a gene by substituting its DNA sequence with that of a genetic marker. Differently, desired mutations can be introduced into a gene by replacing the sequence of the normal gene with that of the mutated gene. Recombinant DNA can be created in vitro and then introduced into cells, most often exploiting the endogenous recombination system of the cells. However, unless the desired mutation gives a particular phenotype, a bottleneck of 'recombineering' is the requirement of a selection system to identify the recombinant clones among those unmodified. Even in an organism like yeast where the level of HR is highly above the incidence of random integration, the frequency of homologous targeting is in the range of 10(-4)-10(-6) depending on the length of the homology used (Wach et al., 1994). Thus, a selection system is always required to identify the targeted clones. Counterselectable markers, such as URA3, LYS2, LYS5, MET15, and TRP1 (Bach and LaCroute, 1972; Chattoo et al., 1979; Singh and Sherman, 1974; Toyn et al., 2000), are widely utilized in yeast and can be recycled for additional usage in the same yeast strain. If the marker is not eliminated or it is popped out via site-specific recombination between direct repeats, such as in the Flp/FRT or Cre/Lox systems, a heterologous sequence is left as a scar at the site of the modified DNA (Storici et al., 1999; Sauer, 1987). The presence of such scars can threaten the genomic stability of the strain and/or limit the number of successive genetic manipulations for that strain. Here, we describe the delitto perfetto approach for in vivo mutagenesis that combines the practicality of a general selection system with the versatility of synthetic oligonucleotides for targeting (Storici et al., 2001). It provides for generation of gene knockouts and almost any sort of mutation and genome rearrangement via HR. The delitto perfetto in vivo mutagenesis technique is designed for efficient and precise manipulation of yeast strains in a two-step process spanning ~2 weeks. Here, we present the theory and procedures of the delitto perfetto technique.

Preferential protection of domains II and III of bacillus thuringiensis cry1Aa toxin by brush border membrane vesicles / Protección preferencial de los dominios II y III de la toxina cry1Aa de bacillus thuringiensis en vesículas de membrana de borde de cepillo

The surface exposed Leucine 371 on loop 2 of domain II, in Cry1Aa toxin, was mutated to Lysine to generate the trypsin-sensitive mutant, L371K. Upon trypsin digestion L371K is cleaved into approximately 37 and 26 kDa fragments. These are separable on SDS-PAGE, but remain as a single molecule of 65 kDa upon purification by liquid chromatography. The larger fragment is domain I and a portion of domain II (amino acid residues 1 to 371). The smaller 26-kDa polypeptide is the remainder of domain II and domain III (amino acids 372 to 609). When the mutant toxin was treated with high dose of M. sexta gut juice both fragments were degraded. However, when incubated with M. sexta BBMV, the 26 kDa fragment (domains II and III) was preferentially protected from gut juice proteases. As previously reported, wild type Cry1Aa toxin was also protected against degradation by gut juice proteases when incubated with M. sexta BBMV. On the contrary, when mouse BBMV was added to the reaction mixture neither Cry1Aa nor L371K toxins showed resistance to M. sexta gut juice proteases and were degraded. Since the whole Cry1Aa toxin and most of the domain II and domain III of L371K are protected from proteases in the presence of BBMV of the target insect, we suggest that the insertion of the toxin into the membrane is complex and involves all three domains.

La superficie de la toxina Cry1Aa, en el asa 2 del dominio II contiene expuesta la leucina 371, la cual fue modificada a lisina produciendo una mutante sensible a la tripsina, L371K. Esta mutante produce dos fragmentos de 37 y 26 kDa por acción de la tripsina que son separables por SDS-PAGE, pero que a la purificación por cromatografía líquida se mantienen como una sola molécula de 65 kDa. El fragmento grande contiene al dominio I y una parte del dominio II (aminoácidos 1 al 371). El polipéptido de 26 kDa contiene la parte restante del dominio II y dominio III (aminoácidos 372 al 609). Cuando la toxina mutante fue tratada con dosis altas de jugo intestinal de Manduca sexta, ambos fragmentos fueron degradados. Sin embargo, cuando fueron incubados en VMBC de M. sexta, el fragmento de 26 kDa fue protegido preferencialmente de las proteasas intestinales. Como se ha reportado, la toxina silvestre Cry1Aa también es protegida de la degradación de las proteasas cuando es incubada en VMBC de M. sexta. Sin embargo, cuando se adicionó VMBC de ratón a la mezcla de reacción, ni la toxina Cry1Aa ni la mutante L371K mostraron resistencia a las proteasas y fueron degradadas. Dado que la toxina completa de Cry1Aa y casi todo de los dominios II y III de L371K están protegidos de proteasas en presencia de VMBC del insecto, este estudio sugiere que la inserción de la toxina en la membrana involucra los tres dominios.