Team members Results & Modeling Lin, Yi-Ting _Department of Molecular Biology and Human Genetics, MBHG Shan, Zhe_MBHG Hsieh, Siang-En _MBHG Chen, Shih-Jie _MBHG Chen, Yu-Ting _MBHG Lien, Pei-Yi_MBHG Hsu, Hao_Department of Medical Informatics We constructed our phagemid with a RFP as reporter gene (shown in Fig.1) and use this to monitor our experiments. The experimental result show phagemid-carrying phage is successfully released (shown in Fig.2 and Fig.3). Then we want to figure out two efficiency question in our experiment ： How to make the most amount of phagemid-carrying phage released, and how to make their infection rate achieve 100%.(Shown as Model A and Model B) Introduction This year, TCU_Taiwan provide a new tool dealing with bacterial antibiotic resistance ---- the Trogene Horse. Our Trogene Horse can produce one-generation phage carrying CRISPR system targeting bacterial antibiotic resistance gene. To achieve this, we use a special phagemid for phage display. The phagemid contains a additional phage replication origin. When helper phage (a phage whose own ori is knocked out) infection takes place, phagemid can be packaged as genome for progeny. Since we put an antibiotic resistance gene targeting CRISPR system on our phagemid, this phage display can spread our CRISPR among bacterium nearby and remove their antibiotic resistance efficiently. Trogene Horse is a new concept combining phage therapy with CRISPR, and this combination can solve both safety concern(phage therapy) and efficiency problem(CRISPR). We believe our project can deal with bacterial resistance problems both in agriculture and medicine. Filamentous phage life cycle Figure 1. Shows that construction of pBluescript with reporter gene RFP. System Design Figure 3. Shows that use ten fold serial dilution before dropping on the plate for six duplicate. Minimized crRNA terminator pRha promoter Tet-off promoter ×1010 T1 terminator pfu/ml tracrRNA Figure 2. Shows that use phage titer determination experiment on agar plate. Phagemid pBluescript II SK(-) M13KO7 Helper phage Incubating time Second structure of package signal Filamentous phage can only infect bacteria with F pili. Its life cycle described in following steps: Adsorption: In this step, M13 and M13KO7 are the same. Both of their pilus attachment protein gp3 and gp6 will help them bind on F pilus, then the phage will move into host cell by F pilus and take off its coat protein. Replication: After infection, both kind of phage’s ssDNA genome will act like a template and use host bacteria’s DNA polymerase to generate replicative form(RF). So the RF can use host’s enzyme to express its genes such as coat protein. Assembly: Then the RF will make gp2 first, this protein can make a nick on intergenic(IG) region of positive strand. A normal M13 phage contains IG region on its f1 ori. But a M13KO7’s f1 ori has been mutated, so gp2 will not work. However, if a phagemid pBluescript, which carries an additional f1 ori, is transformed into the bacteria, gp2 can make a nick on phagemid’s positive strand. The positive strand will then be pulled out and combined with gp5 dimers to stabilize itself. Meanwhile, RF will make coat proteins and other construction proteins, they will move along to host bacteria’s membrane first. Next, gp7 and gp9 will recognize packaging signal around IG (shown in following image) and start packaging. At the same time, gp8 and other proteins will replace gp5 dimers. Finally, a progeny phage is made and released from host bacteria’s membrane. infection time Model A Result from Model A shows that if we incubate bacterium 30 minutes after adding M13KO7 to infect them, and then add kanamycin and incubate for additional 14hr. We will get the best release with amounts of phagemid-carrying phage: 4 x 1010 pfu/ml. DNA replication, Arthur Kornberg, Tania A. Baker, 1991 Shows the intergenic(IG) region of Ff phage. Vertical elements are hairpins in the secondary structure of the viral strand. Team achievements Built and documented biobrick to registry. Experimentally validate Biobricks of our team design and construct were as expected. Use helper phage and phagemid to solve safety problems because those phages cannot have next generation. We improve the function of BBa_K1218011 from 2013 Stanford-Brown . Cooperation with NCTU-Formosa Team on modeling. Human practice Boost synthetic biology to stores near TzuChi university Introduce basic concept of synthetic biology Held iGEM talk at Senior High Schools Promote iGEM and synthetic biology to local senior high school students Attend iGEM conference in NCTU Present our project Communicate ideas with students from Taiwan and China A small iGEM presentation at TzuChi university Introduce the history and format of iGEM Make the 2015 iGEM team get prepared Question Survey Designed Several question based on synthetic biology Video conference with NCTU-Formosa Discussing about modeling part Video conference with NYMU_Taipei Share our project and cheer up each other Acknowledgments Woon, Peng-Yeong, Ching, Yung-Hao, Ingrid Liu, Yen JH. Chen, J.H., Lin, Guang-Huey, Lin, NT., Lin, Ming-Der Model B Result from Model B shows that as long as the MOI is higher than 6 pfu/cfu, the infection rate can achieve almost 100%.
© Copyright 2018