Suspended Liquid Subtractive Lithography: One-step generation of 3D channel geometries in viscous curable polymer matrices

The miniaturization of synthesis, analysis and screening experiments is an important step towards more environmentally friendly chemistry, statistically significant biology and fast and cost-effective medicinal assays. The facile generation of arbitrary 3D channel structures in polymers is pivotal to these techniques. Here we present a method for printing microchannels directly into viscous curable polymer matrices by injecting a surfactant into the uncured material via a steel capillary attached to a 3D printer. We demonstrate this technique using polydimethylsiloxane (PDMS) one of the most widely used polymers for the fabrication of, e. g. microfluidic chips. We show that this technique which we term Suspended Liquid Subtractive Lithography (SLSL) is well suited for printing actuators, T-junctions and complex three dimensional structures. The formation of truly arbitrary channels in 3D could revolutionize the fabrication of miniaturized chips and will find broad application in biology, chemistry and medicine

Inactivation of the Neurospora Crassa Gene Encoding the Mitochondrial Protein Import Receptor Mom19 by the Technique of ``sheltered Rip''

We have used a technique referred to as ``sheltered RIP'' (repeat induced point mutation) to create mutants of the mom-19 gene of Neurospora crassa, which encodes an import receptor for nuclear encoded mitochondrial precursor proteins. Sheltered RIP permits the isolation of a mutant gene in one nucleus, even if that gene is essential for the survival of the organism, by sheltering the nucleus carrying the mutant gene in a heterokaryon with an unaffected nucleus. Furthermore, the nucleus harboring the RIPed gene contains a selectable marker so that it is possible to shift nuclear ratios in the heterokaryons to a state in which the nucleus containing the RIPed gene predominates in cultures grown under selective conditions. This results in a condition where the target gene product should be present at very suboptimal levels and allows the study of the mutant phenotype. One allele of mom-19 generated by this method contains 44 transitions resulting in 18 amino acid substitutions. When the heterokaryon containing this allele was grown under conditions favoring the RIPed nucleus, no MOM19 protein was detectable in the mitochondria of the strain. Homokaryotic strains containing the RIPed allele exhibit a complex and extremely slow growth phenotype suggesting that the product of the mom-19 gene is important in N. crassa

Evidence that a 1.6 kilobase region of Neurospora mtDNA was derived by insertion of part of the LaBelle mitochondrial plasmid.

The LaBelle mitochondrial plasmid hybridizes to a small region of the mtDNA of different Neurospora species. Here, we show that the region of homology encompasses 1385 bp of plasmid sequence and 1649 bp of mtDNA sequence. Several findings--that the region of homology is not found in the mtDNAs of other organisms, that it includes the C-terminus of the ORF encoding the plasmid DNA polymerase, and that the ORF sequence in the mtDNA is interrupted by insertions--suggest that the region was part of the plasmid that integrated into mtDNA prior to the divergence of Neurospora species. Since the LaBelle plasmid has been found in only one Neurospora strain, we infer that the plasmid was lost subsequently from most strains. The LaBelle plasmid is transcribed by the host Neurospora mitochondrial RNA polymerase and the major promoter is located upstream of the long ORF, within the region of homology to mtDNA. A promoter used for the transcription of the mitochondrial small rRNA is found at a corresponding position in Neurospora mtDNA and may have been acquired via integration of the plasmid sequence. Our results provide evidence that an autonomous infectious element may contribute to sequences that functionally constitute an organism's mtDNA