UROP Proceedings 2022-23

School of Engineering Department of Chemical and Biological Engineering 76 The Construction of Dual-functional Synthetic RNA Control Unit Supervisor: KUANG, Becki Yi / CBE Student: HUANG, Qinghua / BIEN Course: UROP1100, Spring In vitro transcription (IVT) is a laboratory technique that enables the production of RNA molecules from a DNA template in a test tube or other container outside of a living cell. In recent years, IVT has become an increasingly popular tool in molecular biology research due to its many advantages over other methods of RNA production. IVT offers high specificity, customizability, scalability, time efficiency, and compatibility with a wide range of downstream applications. To produce EGFP mRNA and iRFP mRNA, we designed and synthesized DNA templates, and subsequently produced mRNAs through IVT with high purity. The IVT protocol consisted of three main steps: 1) producing DNA fragments through PCR; 2) assembling full DNA templates through fusion PCR; and 3) synthesizing RNA through IVT. We used purified DNA templates, ribonucleotide triphosphates, buffer, and phage RNA polymerases to produce RNA in a test tube. The concentration and purity of the DNA and RNA products were checked after each step using Nanoview and gel electrophoresis. Influence of Random Base in Poly(A) Tail to the Function of Mammalian mRNA Supervisor: KUANG, Becki Yi / CBE Student: HUANG, Qinghua / BIEN Course: UROP2100, Summer Different types of proteins, such as transcription factors, receptors, and apoptotic proteins, have been employed in mammalian synthetic biology applications. Traditionally, these protein components are delivered to cells in the form of DNA. However, the use of DNA carries the risk of insertional mutagenesis, which can lead to significant issues in therapeutic contexts. In contrast, synthetic messenger RNAs (mRNAs) offer a promising alternative as they do not pose the same risk of insertional mutagenesis. Furthermore, synthetic mRNAs incorporating modified nucleosides can effectively evade the induction of inflammation caused by nucleic acids. In our program, we make use of a novel tool called Caliciviral VPg-based Translational activator (CaVT) and mRNA Decapping activators Scd6 combining with engineered pairs of distinct photoswitches to construct a light control mRNA translation activator and deactivator. Design and Construction of Genetic Landing Pad in Probiotic Bacteria Supervisor: LAI, Yong / CBE Student: YU, Kai Lam Karl / SENG Course: UROP1100, Summer Advances in synthetic biology are driven by gene circuitry. In order to increase the stability and reliability of genetic circuits in bacteria over time and reduce the genetic burden on the host cell, the circuits should be integrated into the genome. However, genetic manipulation of probiotic bacteria is at an early stage. The progress in developing genome-editing tools is also constrained by limitations in the availability of plasmids. Here, we will develop novel genome engineering tools in our bacterial chassis and integrate validated genetic circuits into their genomes to ensure long-term, stable functionality.