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We present a new concept in DNA engineering based on a pipeline of serial recombineering steps in liquid culture. This approach is fast, straightforward and facilitates simultaneous processing of multiple samples in parallel. We validated the approach by generating green fluorescent protein (GFP)-tagged transgenes from Caenorhabditis briggsae genomic clones in a multistep pipeline that takes only 4 d. The transgenes were engineered with minimal disturbance to the natural genomic context so that the correct level and pattern of expression will be secured after transgenesis. An example transgene for the C. briggsae ortholog of lin-59 was used for ballistic transformation in Caenorhabditis elegans. We show that the cross-species transgene is correctly expressed and rescues RNA interference (RNAi)-mediated knockdown of the endogenous C. elegans gene. The strategy that we describe adapts the power of recombineering in Escherichia coli for fluent DNA engineering to a format that can be directly scaled up for genomic projects.

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Even though motion planning for automated vehicles has been extensively discussed for more than two decades, it is still a highly active field of research with a variety of different approaches having been published in the recent years. When considering the market introduction of SAE Level 3+ vehicles, the topic of motion planning will most likely be subject to even more detailed discussions between safety and user acceptance. This paper shall discuss parameters of the motion planning problem and requirements to an environment model. The focus is put on the representation of different types of uncertainty at the example of sensor occlusion, arguing the importance of a well-defined interface between decision making and trajectory generation.