Difference between revisions of "Team:Kyoto/Description"

Line 4: Line 4:
 
<style>
 
<style>
  
  body{
 
    margin: 0;
 
  }
 
 
   #background{
 
   #background{
 
     position:fixed;
 
     position:fixed;

Revision as of 08:37, 18 October 2016

Project(文責:中村・山田)




1. Abstract

Norovirus (NoV) accounts for 18% of the diarrheal disease in the world, with more than 200 thousand people dying each year from NoV infection. [1] Despite this overwhelming damage caused by NoV infection, the world has yet to develop a direct approach to combat them. Here we, iGEMKyoto, propose ‘Noro-catcher’, a new biodevice that binds to and ultimately expels NoV from human intestine. This biodevice consists of two types of functional handles that are each fused to surface expressing domains, both expressed in the outer membrane of E. coli.

The first handle is the anti-NoV scFv (single chain variable fragment). With this handle, we aim our Noro-catcher to bind to NoV within various environments, e.g. sewage, water supplies, test tubes in virus detection assay, and small intestines in our body.

The second handle is the cellulose binding domain (CBD). With this handle, we aim our Noro-catcher to be “leashed” to cellulose as opposed to it being “free roaming” within human digestive tracts. We ultimately aim to use our Noro-catcher for therapeutic purposes, removing E.coli-bound NoV from human intestine with our sterilized Noro-catcher. The CBD handle can aid in swift removal of our biodevice by linking them to cellulose, as cellulose passes human intestine undigested.

To express these two handles in the outer membrane region of E.coli, we enhanced upon a surface expression protein domain called INPNC, and used it as the anchor. With scanning electron microscopy, we observed the binding of our Noro-catcher (only with the surface expressing scFv handle) to NoV-like particles, which is the capsid proteins of NoV. With fluorescent microscopy, we have also observed the binding of our Noro-catcher (only with the surface expressing CBD handle) to cellulose.

We demonstrated each of the noro-catcher handles’ functionality by physically binding them to NoVLP and cellulose. As shown by our project, creating a recombinant bacteria expressing combination of target-binding modules dramatically enhances each modules’potentials. This mechanism can be applied to removal system of other harmful agents, including other pathogens, organic toxins, and heavy metals.

2. Introduction

2-1 Global burdens of NoV infection and progress of its countermeasures

Norovirus (NoV)causes inflammation of the stomach and/or intestines, which is called gastroenteritis. When infected with NoV, a person usually develops symptoms in 12 to 48 hours, and most will get better within 3 days. [2][3] However, when its symptoms worsens, it can lead to death. Its common symptoms include diarrhea, vomiting, nausea, and severe stomach pain. Fig1,2 shows the recent prevalence of NoV infection among the world.

The data for U.S. and U.K. seems relatively low (Fig1), but NoV cannot be ignored in those developed countries. As shown in Fig2, NoV remains a significant issue to overcome in both developed and developing countries.

Fig.1 The yearly number of NoV patients per population of 100,000. The data indicates more than 10 in 1 people of Africa are infected by NoV annually. [16][17][18]
Fig.2 The percentage of food poisoning caused by NoV amongst all food poisoning cases in U.S. and U.K. Almost half of all food poisoning are caused by NoV in the two countries. [17][18]

There are treatments and drugs to alleviate individual symptoms like vomiting and nausea. However, there are none that targets NoV itself. As NoV's infection mechanisms and its cultivation condition was not known until recently, research on such methods were significantly hindered. [13] We therein resolved to devote our knowledge and skills of synthetic biology to join the global fight against the unconquered and highly contagious NoV.

Recent studies show that NoV seems to be multiplying in the human B cells, binding to the Histo-blood group antigen (HBGA) of enteric cells and red blood cells to get within the intestinal epithelial barrier [3] [5] [12]. Another studies show that human anti-NoV antibodies, such as antibody12A2, binds to NoV’s HBGA-binding site. [19] It suggests that the antibody sterically hinders NoV from binding to HBGA and blocks NoV’s entry into human body, effectively neutralizing them (Fig3).

Fig3. Infection route of NoV and neutralization mechanisms of 12A2 antibody.
NoV binds to the red blood cell's HBGA to pass through intestinal epithelial cells to multiply in the B cells. Human 12A2 antibody sterically hinders NoV's binding to HBGA. [3] [5] [12] [19]
Reference [1]Infectious Diseases of the World (GIDEON Informatics, Inc.、Dr. Stephen Berger) [2]Morillo, Simone Guadagnucci, and Maria do Carmo Sampaio Tavares Timenetsky. "Norovirus: an overview." Revista da Associação Médica Brasileira 57.4 (2011): 462-467. [3]Karst SM, Wobus CE (2015) A Working Model of How Noroviruses Infect the Intestine. PLoS Pathog11(2):e1004626.doi:10.1371/journal. Ppat.1004626 [4]Ettayebi, Khalil, et al. "Replication of human noroviruses in stem cell–derived human enteroids." Science 353.6306 (2016): 1387-1393. [5]Jones, Melissa K., et al. "Enteric bacteria promote human and mouse norovirus infection of B cells." Science 346.6210 (2014): 755-759. [6]Kawahara, Hidehisa. "The structures and functions of ice crystal-controlling proteins from bacteria." Journal of bioscience and bioengineering 94.6 (2002): 492-496. [7]Park, Tae Jung, et al. "Surface display of recombinant proteins on Escherichia coli by BclA exosporium of Bacillus anthracis." Microbial cell factories 12.1 (2013): 1. [8]Tomme, Peter, et al. "Characterization and affinity applications of cellulose-binding domains." Journal of Chromatography B: Biomedical Sciences and Applications 715.1 (1998): 283-296. [9]It is iGEM Bielefeld 2012 that have made the CBDcex and CBDclos into Biobrick parts, and iGEM Imperial 2014 added on to these parts by fusing them with sf-GFP. Since the parts by iGEM Imperial 2014 was included in the kit, we have chosen to use theirs for the construction. For more information about the parts, please visit their respective pages. [10]https://2014.igem.org/Team:WPI-Worcester/Proof-of-Principle [11]Shanker, Sreejesh, et al. "Structural basis for norovirus neutralization by an HBGA blocking human IgA antibody." Proceedings of the National Academy of Sciences (2016): 201609990. [12]Ettayebi, Khalil, et al. "Replication of human noroviruses in stem cell–derived human enteroids." Science 353.6306 (2016): 1387-1393. [13]This summer, a team of scientists reported a way to incubate NoV using human stem cells, and research on these fields are expected to progress rapidly.[4] However, it still remains the case that a definitive method against NoV infection are nowhere to be found. [*]MacLeod, Alasdair M., et al. "Mechanistic consequences of mutation of active site carboxylates in a retaining β-1, 4-glycanase from Cellulomonas fimi." Biochemistry 35.40 (1996): 13165-13172. [14]Hardy, Michele E. "Norovirus protein structure and function." FEMS microbiology letters 253.1 (2005): 1-8. [15] Wang, Aijun A., Ashok Mulchandani, and Wilfred Chen. "Specific adhesion to cellulose and hydrolysis of organophosphate nerve agents by a genetically engineered Escherichia coli strain with a surface-expressed cellulose-binding domain and organophosphorus hydrolase." Applied and environmental microbiology 68.4 (2002): 1684-1689. [16]Mans, Janet, et al. "Norovirus Epidemiology in Africa: A Review." PloS one 11.4 (2016): e0146280. [17] Food Standards Agency (2012): Annual Report of the Chief Scientist https://www.food.gov.uk/sites/default/files/multimedia/pdfs/publication/csar1112.pdf [18] Centers of Disease Control and Prevention (2016): Estimates of Foodborne Illness in the United Statesn https://www.cdc.gov/foodborneburden/burden/index.html [19] Kyoko Higo-Moriguchi, Haruko Shirato, Yuichi Someya, Yoshikazu Kurosawa, Naokazu Takeda, and Koki Taniguchi. “Isolation of Cross-Reactive Human Monoclonal Antibodies That Prevent Binding of Human Noroviruses to Histo-Blood Group Antigens” Journal of Medical Virology 86:558–567 (2014) [20] In general, purification and characterization of proteins require it to be soluble. Insoluble samples need to be denatured and solubilized to be handled, but proteins would completely lose its charasteristics and functions, disabling purification. However, His tag's unique ability to retain its property to bind to nickel after denaturing means it can exceptionally be purified even when in insoluble sample.