Introduction of Laboratory Animal Resources Bank (LAREB) Kyung-Ku Kang , Min-Soo Seo , Soo-Eun Sung , Joo-Hee Choi¹, SI-Joon Lee¹, 1 1 1 Min-Kyung Sung¹, Kil-Soo Kim * 1 1 Laboratory animal center, Daegu Gyeongbuk Medical Innovation Foundation, Daegu, Korea Abstract Laboratory animal Resources Bank (LAREB) is national infrastructure from National Institute of Food and Drug Safety Evaluation (NIFDS, Korea) to utilize resources of laboratory animals that have been experimentally intervened. After finishing the research, collecting resources from disused laboratory animals and sharing them with
other researchers will make the national infrastructure to reduce the research cost and shorten the research period. LAREB receives donated resources through the core facility (DGMIF, Korea) which produces resources through primary processing from laboratory animals received from universities, research institutes and pharmaceutical companies. Main resources are rare laboratory animals, long-term drug treated animals, rare substance treated animals and animals that have undergone difficult surgery. Donated resources are divided by organ and stored in formalin fixed, paraffin block, hematoxylin
& eosin slide and frozen tissue. LAREB has stored about 30,000 resources to date, the largest proportion of which is frozen tissue resources that can generate secondary processing resources such as RNA and protein. Therefore, we think that it will can vitalize donation and utilization of resources through various and continuous publicity of LAREB. By operating the LAREB, we expect to strengthen research competitiveness, create diverse results from one study, and create new industries for high value-added laboratory animals and resources. Laboratory Animal Resources Bank (LAREB) Resource type
stored in LAREB LAREB DGMIF (Core facility) LAREB Comparison of workflow Resource Resource Donation Core facility Utilization University Laboratory Company Resource Processing & Storing System Barcode, Database Processing Fixed tissue Paraffin block H&E Slide Resource Donation Necropsy Tissue Frozen Tissue [C. Biotechnology and molecular imaging-2] Introduction of Laboratory Animal Resources Bank (LAREB) Kyung-Ku Kang¹, Min-Soo Seo¹, Soo-Eun Sung¹, Joo-Hee Choi¹, SI-Joon Lee¹, Min-Kyung Sung¹, Kil-Soo Kim¹ ¹Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), Daegu
41061, Korea Laboratory animal Resources Bank (LAREB) is national infrastructure from National Institute of Food and Drug Safety Evaluation (NIFDS, Korea) to utilize resources of laboratory animals that have been experimentally intervened. After finishing the research, collecting resources from disused laboratory animals and sharing them with other researchers will make the national infrastructure to reduce the research cost and shorten the research period. LAREB receives donated and distributes resources through the core facility (DGMIF, Korea). Main resources are rare laboratory animals,
long-term drug treated animals, rare substance treated animals and animals that have undergone difficult surgery. Donated resources can be classified several resource types such as formalin fixed tissue, paraffin block, H&E slide and frozen tissue which called bio-resources from laboratory animals. LAREB provides resource information through online and the researcher searches for needed resources and requests a distribution. By operating the LAREB, we expect to strengthen research competitiveness, create diverse results with one study, create new industries for high value-added laboratory
animals and bio-resources. And we hope to create new research environment for using of ethical laboratory animals. Auto-inducible, dynamic, and antibiotic-free expression system in Escherichia coli for the production of farnesol 1 Mst. Fayzunnesa , and Seon-Won Kim 1 1 Division of Applied Life Science (BK21 plus Program), PMBBRC, Gyeongsang National University, Jinju 52828, Republic of Korea Background AIM Engineered E.coli MG1655 strain overexpressing heterologous MVA The main purpose of this study is to pathway genes or along with native MEP, pathway produces IPP and establish an
auto-inducible and dynamic DMAPP buildingblocks for FPP synthesis catalyzed by FPP synthase system in E.coli for the production of (IspA). Subsequently, FPP could then be depyrophosphorylated to Farnesol in an economic way. form farnesol via NudJ, an E.coli native phosphatase suspected to dephosphorylate IPP and DMAPP to isoprenoid alcohols. To establish Secondly, the purpose of the study is to an auto-inducible and dynamic system IspA, idi, and MVA pathway introduce an antibiotic-free bio-culture genes were overexpressed under the control of inducer-free promoters system in E. coli for the
production of PgadE and PrstA successfully producing the targeted products farnesol on a large scale and also accost farnesol for 48 hours. effective manner. METHODS Construction of these following plasmid and then culture them for production RESULTS Figure 1. Cell growth and Production improvement by Figure 2 Large scale production of Farnesol using an antibiotic-free plasmid-based system comparing with using in E. coli MG1655 with antibiotic and an inducer-free system in E. coli MG1655. inducible plasmid based system comparing antibiotic-free system CONCLUSION REFERENCES ACKNOWLEDGEMENTS
Farnesol can be a very [1] Wang, Chonglong, et al. "Metabolic This work was supported by a grant promosing candidate for engineering and synthetic biology approaches from the Next-Generation BioGreen 21 advanced biofuel and driving isoprenoid production in Escherichia Program (SSAC, grant#: PJ01326501), commodity chemicals. coli." Bioresource technology 241 (2017): 430- Rural Development Administration, Republic of Korea. 438. Introducing Some inducer-free [2] Zada, Bakht, et al. "Metabolic engineering of and Antibiotic-free expression Escherichia coli for production of mixed Contact
information system farnesol can be isoprenoid alcohols and their produced in a successful way. derivatives." Biotechnology for biofuels 11.1 E-mail: swkim@gnu.ac.kr (2018): 210. E-mail: mst.fayzunnesa@yahoo.com . [C. Biotechnology and molecular imaging-4] Auto-inducible, dynamic, and antibiotic-free expression system in Escherichia coli for the production of farnesol Mst Fayzunnesa¹, Seon-won Kim¹˙* ¹Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju 52828, Republic of Korea Engineered E.coli MG1655 strain overexpressing heterologous MVA pathway
genes or along with native MEP, pathway produces IPP and DMAPP building blocks for FPP synthesis catalyzed by FPP synthase (IspA). Subsequently, FPP could then be depyrophosphorylated to form farnesol via NudJ, an E.coli native phosphatase suspected to dephosphorylate IPP and DMAPP to isoprenoid alcohols. To establish an auto-inducible and dynamic system IspA, idi, and MVA pathway genes were overexpressed under the control of inducer-free promoters PgadE and PrstA instead of inducer dependent promoter Plac and Ptrc successfully producing 384 mg/L total farnesol and farnesol derivatives
(farnesal and farnesyl acetate) for 48 hours. The development of sustainable large scale farnesol production requires an antibiotic-free system in order to avoid additional downstream purification and separation steps while decreasing overall production cost. The antibiotic-free strain was able to maintain the production plasmids without antibiotics, achieving 1.4g/L of farnesol and farnesol derivatives in a fed-batch culture system or 1.3fold better than with antibiotics. This work was supported by the grant from the Next-Generation BioGreen 21 Program (SSAC grant#: PJ01326501), RDA, Korea.
Antibiotic-free system for phytoene production from recombinant E. coli Seung-Hye Lee , Seon-Won Kim * 1 1 1 Division of Applied Life Science (BK21 plus Program), PMBBRC, Gyeongsang National University, Jinju 52828, Republic of Korea *Corresponding author: Prof. Seon-Won Kim, Tel.: 82-55-772-1362; Fax: 82-55-759-9363; E-mail address: swkim@gnu.ac.kr BACKGROUND AIM Phytoene is colorless and absorbs UV-B light from 260 to In this study, for the improved production of phytoene, 320 nm which can be used as a photo-protective material we applied an antibiotic-free expression system. The to prevent
urban skin aging. In order to synthesize system is based on the isoprenoid pathway such as phytoene in E. coli, the phytoene biosynthesis pathway is the MEP pathway and the MVA pathway. These introduced by expression vectors. Expression vectors are pathways are required for the production of IPP and maintained by antibiotics. However, when antibiotics are DMAPP which are essential metabolites for microbial used in the fermentation process, it causes increase in growth. The genes consisting of the pathways are production costs. Moreover, as the culture progresses for used instead of the
antibiotic marker gene applying a long time, the antibiotics are decomposing and the selection pressure to maintain the plasmids.. plasmid disappears, leading to a decrease in production. METHODS Culture was carried out for 72 hours in 2YT medium containing 3.0%(w/v) glycerol, 0.2%(w/v) arabinose at 30℃, +with antibiotics, - without antibiotics. Ampicillin and chloramphenicol were added as needed as antibiotics used, and were used at concentrations of 100µg/mL and 50µg/mL respectively. Phytoene was extracted using acetone. The extract was centrifuged at 14,000 rpm for 10 minutes, and only the
top solution was taken to conduct a quantitative HPLC analysis. RESULTS Table 1. Phytoene production strain. Figure 2. Plasmid stability test of Antibiotic-free Host Strain 1 Plasmid 2 nd Plasmid phytoene production system. st MG1655(DE3) pSNA(-E) pT-PHYm3coE C-AF pla AF Host MG1655(DE3)ΔdxrΔadhE::MVAbottom pSNA pT-PHYm3co AF MG1655(DE3)ΔdxrΔadhE::MVAbottom pSNA(-E) pT-PHYm3coE dxr is one of the genes in the MEP pathways of E. coli.. pSNA(-E), MVA pathway expression vector except mvaE; pT-Phym3coE, phytoene Plasmid Stability (%) = production plasmid containing mvaE. on Antibiotics containing
plate X 100(%) Figure 1. Phytoene production in antibiotic-free on Antibiotics containing plate recombinant E. coli. Antibiotics + - + - + - 1200 24 Hr 100 48 Hr 72 Hr 1000 80 Phytoene (mg/L) 600 Plasmid Stability (%) 60 800 40 400 200 20 0 0 0 24 48 72 C-AF pla AF Host AF Time(hr) CONCLUSION REFERENCES ACKNOWLEDGEMENTS As a result, it is possible to maintain the [1] Antonio J.Meléndez-MartínezPaulaMapelli- This work was supported by a grant plasmid without antibiotics and obtain BrahmCarla M.Stinco 2018. The colourless from the Next-Generation BioGreen 21 improved production of phytoene With
carotenoids phytoene and phytofluene: From Program (SSAC, grant#: PJ01326501), or without antibiotic, the plasmid dietary sources to their usefulness for the Rural Development Administration, remained at 100% for up to 72 hours. functional foods and nutricosmetics industries. Republic of Korea. J. Food Anal., 67 (2018), pp. 91-103 This confirmed the establishment of an [2] Peubez., I., Chaudet, N., Mignon, C., Hild, antibiotic-free phytoene production G., Husson, S., Courtois, V., et al. Antibiotic- Contact information system and it was confirmed that even free selection in E. coli: new
considerations for when the incubation time is prolonged optimal design and improved production. Micrb. Tel.: 82-55-772-1362 the plasmid can be maintained stably. Cell Fact. 9 2010, 65. doi: 10.1186/1475-2859- Fax: 82-55-759-9363 9-65 E-mail address: swkim@gnu.ac.kr [C. Biotechnology and molecular imaging-5] Antibiotic-free system for phytoene production from recombinant E. coli Seung-Hye Lee¹, Seon-Won Kim¹˙* ¹Division of Applied Life Science (BK21 Plus Program), PMBBRC, Gyeongsang National University, Jinju 52828, Republic of Korea Phytoene is colorless and absorbs UV-B light from 260 to 320
nm which can be used as a photo-protective material to
