Deletion of the Pichia pastoris KU70 Homologue Facilitates Platform Strain Generation for Gene Expression and Synthetic Biology

Targeted gene replacement to generate knock-outs and knock-ins is a commonly used method to study the function of unknown genes. In the methylotrophic yeast Pichia pastoris, the importance of specific gene targeting has increased since the genome sequencing projects of the most commonly used strains have been accomplished, but rapid progress in the field has been impeded by inefficient mechanisms for accurate integration. To improve gene targeting efficiency in P. pastoris, we identified and deleted the P. pastoris KU70 homologue. We observed a substantial increase in the targeting efficiency using the two commonly known and used integration loci HIS4 and ADE1, reaching over 90% targeting efficiencies with only 250-bp flanking homologous DNA. Although the ku70 deletion strain was noted to be more sensitive to UV rays than the corresponding wild-type strain, no lethality, severe growth retardation or loss of gene copy numbers could be detected during repetitive rounds of cultivation and induction of heterologous protein production. Furthermore, we demonstrated the use of the ku70 deletion strain for fast and simple screening of genes in the search of new auxotrophic markers by targeting dihydroxyacetone synthase and glycerol kinase genes. Precise knock-out strains for the well-known P. pastoris AOX1, ARG4 and HIS4 genes and a whole series of expression vectors were generated based on the wild-type platform strain, providing a broad spectrum of precise tools for both intracellular and secreted production of heterologous proteins utilizing various selection markers and integration strategies for targeted or random integration of single and multiple genes. The simplicity of targeted integration in the ku70 deletion strain will further support protein production strain generation and synthetic biology using P. pastoris strains as platform hosts

Bituminous binder

One of the most important aspects of asphalt pavement deterioration is the ingress of water in pavement which leads to loss of the material characteristics, even material integrity with loss of aggregates. Thus the behaviour of asphalt mixture under moisture conditions is one of the key parameter for specifications. It’s a complex phenomenon which is influenced amongst other things by materials properties with wetting, cohesion and adhesion of bituminous binder and by environmental conditions with temperature, moisture, loading and layer type. It has been a research subject for a very long time and still not precisely described. A large number of test methods is available to estimate the affinity between aggregates and bituminous binders. These test methods can be subdivided in different ways; a first distinction can be based on the presence or absence of water during the test procedure. If water is present, the evaluation is in fact referred to as water sensitivity or moisture damage testing. Another distinction can be based on the type of sample that is evaluated. The test sample can be loose aggregates coated with a bituminous binder or a compacted asphalt mix sample. Lastly the individual components, bitumen and aggregate, can be tested separately through intrinsic properties. Furthermore, test results can also be based on the quantification of the test results, whether this is based on a qualitative or a quantitative evaluation. In RILEM TC 237 SIB, TG1 the main purpose was to evaluate common test methods, used to assess the affinity of bitumen to aggregate surfaces, to determine, if possible, the repeatability and reproducibility and to give recommendations for improvement. In this study both binders and aggregates have been considered. Three bituminous binders, two unmodified from different sources, one polymer modified binder, and four aggregate types, with different mineralogy, have been selected. The test methods considered in the study include the rolling bottle test, the boiling water stripping test and the bitumen bond strength test; also surface energy was investigated. This chapter presents the results of these tests and their accuracy