School of Science Division of Life Science 33 Cloning and Characterization of Novel Cytoskeletal Regulators Supervisor: QI Robert Zhong / LIFS Student: PARK Joo Hyoung / BIOT Course: UROP2100, Spring CRISPR is a novel genetic engineering technology widely applied to many areas of biochemistry and cell biology, originating from the naturally occurring adaptive immunity response system in bacteria. Its uses involve knockout of genes, activation/repression of genes, purification of DNA, precise editing of DNA and RNA, and more. This report discusses the use of this technique with applied assays such as microtubule regrowth assay and FACS assay to be able to identify and study in details the genes responsible for the nucleation of microtubules at MTOCs, with additional insight into how exactly CRISPR could be applied and focusing particularly on plasmids including psPAX-2, pMD-2G, pBABE-ZEO. Cloning and Characterization of Novel Cytoskeletal Regulators Supervisor: QI Robert Zhong / LIFS Student: CATIELLO Carlo / BIOT Course: UROP1000, Summer The γ-tubulin ring complex (γ-TuRC) is a protein complex composed of γ-tubulin small complexes (γTuSC) and multiple γ-tubulin complex proteins (GCP) that regulates microtubule nucleation by providing control over microtubule growth. Neural precursor cell expressed developmentally down-regulated protein 1 (NEDD1) is an important component of the centrosome that directly interacts with γ-tubulin and targets it to the chromosome. In this experiment, conventional molecular cloning methods were used to clone NEDD1 579-667 to utilize it as a bait to pull down γ-TuRC-bound NEDD1 from the centrosomes. A mutated version of the protein, NEDD1 579-667 H615F, which have reduced binding ability to γ-tubulin was also cloned to be used as a control. Confirmation of the experiment was done by double digestion as well as DNA sequencing. Investigation of Novel Mechanism Underlying Microtubule Organization Supervisor: QI Robert Zhong / LIFS Student: AU Chun Kei / CIVL Course: UROP1000, Summer The γ-tubulin plays an important role in microtubule organization. Under normal situations, microtubule is made up of both lateral and longitudinal γ-tubulin interactions by polymerization. In this research study, our goal is to detect the lateral binding acid also known as γ-tubulin-4A-GFP. It is known that the mutant protein γ-tubulin-4A will disrupt the longitudinal γ-tubulin interaction. Therefore, when we construct the γ-tubulin4A mutant, it will disrupt the γ-tubulin/ -tubulin and γ-tubulin/γ-tubulin longitudinal interaction leaving only the lateral γ-tubulin interactions. So, the detection of lateral γ-tubulin interactions is crucial for investigating the γ-tubulin-4A mutant protein. To aid our investigation on the mutant protein and its construct, we need to carry out several experimental procedures.