Engineering Enzyme Specificity Using Computational Design of a Defined-Sequence Library

Engineered biosynthetic pathways have the potential to produce high-value molecules from inexpensive feedstocks, but a key limitation is engineering enzymes with high activity and specificity for new reactions. Here, we developed a method for combining structure-based computational protein design with library-based enzyme screening, in which inter-residue correlations favored by the design are encoded into a defined-sequence library. We validated this approach by engineering a glucose 6-oxidase enzyme for use in a proposed pathway to convert D-glucose into D-glucaric acid. The most active variant, identified after only one round of diversification and screening of only 10,000 wells, is approximately 400-fold more active on glucose than is the wild-type enzyme. We anticipate that this strategy will be broadly applicable to the discovery of new enzymes for engineered biological pathways.United States. Office of Naval Research. Young Investigator Program (Grant N000140510656)National Science Foundation (U.S.) (Synthetic Biology Engineering Research Center. Grant EEC-0540879)MIT Faculty Start-up FundCodon Devices, Inc

Creation of a type IIS restriction endonuclease with a long recognition sequence

Type IIS restriction endonucleases cleave DNA outside their recognition sequences, and are therefore particularly useful in the assembly of DNA from smaller fragments. A limitation of type IIS restriction endonucleases in assembly of long DNA sequences is the relative abundance of their target sites. To facilitate ligation-based assembly of extremely long pieces of DNA, we have engineered a new type IIS restriction endonuclease that combines the specificity of the homing endonuclease I-SceI with the type IIS cleavage pattern of FokI. We linked a non-cleaving mutant of I-SceI, which conveys to the chimeric enzyme its specificity for an 18-bp DNA sequence, to the catalytic domain of FokI, which cuts DNA at a defined site outside the target site. Whereas previously described chimeric endonucleases do not produce type IIS-like precise DNA overhangs suitable for ligation, our chimeric endonuclease cleaves double-stranded DNA exactly 2 and 6 nt from the target site to generate homogeneous, 5′, four-base overhangs, which can be ligated with 90% fidelity. We anticipate that these enzymes will be particularly useful in manipulation of DNA fragments larger than a thousand bases, which are very likely to contain target sites for all natural type IIS restriction endonucleases

The effects of whole body vibration on lower extremity muscle performance - systematic literature review

Uvod: Vibracije celotnega telesa so mehanski dražljaj, ki vstopi v človeško telo skozi stopala medtem ko oseba stoji na vibracijski plošči v različnih položajih ali izvaja vaje. Ker obstajajo nasprotja glede koristnosti vibracij celotnega telesa in njenih optimalnih parametrov, je bil namen pregleda ugotoviti vpliv vadbe z vibracijami celotnega telesa na mišično zmogljivost spodnjega uda. Metode: Pregledana je bila podatkovna zbirka PubMed, iz katere so bile na podlagi postavljenih meril izbrane raziskave za vključitev v pregled. Rezultati: Merilom je ustrezalo 15 raziskav, v katerih so primerjali učinkovitost vadbe z vibracijami celotnega telesa z enako vadbo, ki jo je kontrolna skupina izvajala brez vibracij. V devetih raziskavah so ugotovili, da je vadba z vibracijami pri izboljšanju posameznih komponent mišične zmogljivosti učinkovitejša kot vadba, izvedena brez vibracij. V preostalih so ugotovili, da vibracije celotnega telesa nimajo dodatnega učinka. Zaključki: Vibracijska vadba ni le primerljiva z vadbo brez vibracij, temveč ima tudi dodatno korist pri izboljševanju mišične zmogljivosti, predvsem komponente maksimalne izometrične mišične sile. Različni vadbeni programi imajo različne vadbene učinke, vendar zaradi prevelikih razlik v uporabljenih metodologijah vibracijske vadbe optimalnih parametrov vibracij in vadbenega protokola ni bilo mogoče natančno določiti.Background: Whole-body vibration (WBV) represents the mechanical stimulus that enters the human body through the feet, while a person is standing on a vibrating plate in different positions or performing exercises. Because of the existing contradictions as to whether WBV is beneficial and which parameters may be the most suitable, the purpose of the review was to determine the effect of WBV training on lower extremity muscle performance. Methods: Literature search was performed on the PubMed database from where, based on the set criteria, studies were selected to be included in the review. Results: 15 studies in which they compared the effectiveness of vibration exercise with the same exercise performed without vibration met the criteria. In nine studies, they found that the exercise with vibration is more effective in improving individual components of muscular performance than the exercise without vibration. The rest found no additional effect of WBV. Conclusions: Vibration exercise is not only comparable to exercise without vibration, but also has an additional benefit in improving muscle performance, particularly isometric muscle strength. Different protocols have different training effects, but because of many differences in the methodologies used for vibration training, optimal vibration characteristics and exercise protocol could not be accurately determined