Difference between revisions of "Team:RDFZ-China/Human Practices"

Probiotic Production

Probiotics, by definition, are living microorganisms which when administered in adequate amounts confer a health benefit on the host. It has long been suggested to be health beneficial with effects like prevention of AAD (antibiotic associated diarrhoea)[1], increase HDL in blood[2], and when prescription of antibiotic has interrupt microbial balance in the gastrointestinal tract, probiotic may be able to restored the balance[1]. Nowadays, people have increased awareness of probiotics’ importance for health, the demand from the market thus rise and various production techniques are employed. In order for the probiotics to maintain their effectiveness all though their shelf-life, they need to be dried into pallets or powder before releasing into the market. Spray, freeze, vacuum and fluid bed drying are four major ways[3]. However, stress would be induced during the drying process and will severely affect the viability and thus the effectiveness of probiotics after rehydration.

Different drying techniques brought different stress

Within all these different methods, they have one thing in common—various stress are induced during the dehydration process, examples like shear stress [4,8], oxidative stress[5,8], heat stress[6,8], chemical stress and osmotic stress[7,8]. Cell membrane in particular, appears to be damaged and significantly affect cells viability. Its fluid bilayer structure was originally stabilised via Van der Waals forces and hydration repulsion, which is severely weakened by lose of water[7] .Package default will occur during rehydration process because the regular arrangement of phospholipids is disrupted during dehydration, and this cause leakage of cellular components[9]. Dehydration brought severe decrease in viability of the cells after rehydration and cells’ resilience seems to be a major concern.

Protection by adding protective agent

Three main protective strategies can be envisaged to maintain their Viability after dehydration:

1. Change the process parameters, like lowering the temperature, or pressure within certain range [10]


2. Pre-stressing the cell prior to drying, which induces their own protective stress responses [11]


3. Add protective agents.

Saccharides(e.g. Sucrose, lactose, trehalose) [12,13]is one of the most commonly used protectant and was proved to be efficient in improving resilience of cells, several correlated mechanisms have been proposed. Trehalose, a non-reducing disaccharide of glucose, have been widely accepted as a long-time protectant. However, cost of production is significantly increased by artificially adding trehalose, and we are hoping to find a suitable substituent for it.

The phylum Tardigrada

They roamed the island and sea that human has never been, they survived though 5 masses extinction and given rise to about 1150 knowned species[17]. These strangely cute organisms, with cylindrical body and stubby eight legs are famous for their ability to survive in extreme conditions that would be fatal to other life forms, phrases like “toughest organism” “return from the dead” are frequently seen together with their names. Because of their peculiar resilience, their traces could be found almost everywhere, across the seven continent including Antarctica, a wide range of biomes, the dessert, ice field, deep sea or maybe in your garden. They are able to tolerate deadily UV radiation and osmotic rays[18], as well as the almost complete dehydration by switching to ‘anhydrobiotic state’, which confer resistance towards various environmental extremes.

What’s worth mentioning is that, anhydrobiotic ability is observed in various invertebrates, tardigrades, arthropods, and nematodes[14]. Trehalose are suggested to have an important role mediating desiccation resistance in anhydrobiotic organisms, however, the accumulation of trehalose is observed when the last two phylum subjected to desiccation, but not in tardigrades[15,16]. With further searching, we noticed that tardigrades’ anhydrobiotic ability are mediated by a rather novel class of protein called TDPs (tardigrades intrinsically disorded proteins)----the protein that our iGEM team is currently focusing on.

About our project

a) Inspiration

Every year, iGEM competition motivates teams from all over the world to devise numerous great project, genetically engineered organisms are designed to serve in wide range of fields. However, when it comes to application, the regulation of gene expression is not the only rising issue, but also the resilience of these engineered organisms that we need to concern. For example, some team’s bacteria have to work in dessert with extremely low water content(.ref), or when cell components are freeze dried on the test paper to make a paper based biosensor, the system must undergo severe dehydration for storage and transport, and this would probably hamper their effectiveness during work.(.ref). Our investigationfor the 2016 iGEM projects showed that over 299 entries, x% engineered organisms are facing practical issues related with extreme working conditions or environmental stress. As a consequence, team SIAT-SCIE is focusing on how we can transform the resilience of tardigrades into the engineered organisms. Increasing their efficiency during work and gives them greater potential to be put into real practice.

b) What are we doing?

1. Our first goal is to test whether TDPs can provide desiccation resistance for enzyme in vitro.


2. Secondly, we transfer our designed plasmid into DH5α and express TDP in vivo to see if it can confer desiccation resistance, by comparing the engineered strain with the E. coli that didn’t express the TDP gene. Hence a protection mechanism is developed, which can facilitate other iGEM teams engineered organisms to work efficiently.


3. During ametabolic state, the DNA repairing mechanism is halted and cells are vulnerable to mutagenic radiation. Hence our final goal is to ameliorate our protection system by expressing the protein Dsup, in providing resistance towards radiation for the engineered bacteria, as well as MnSOD, which protect against oxidative stress during desiccation process.

Reference:

1. Goldenberg JZ, Lytvyn L, Steurich J, Parkin P, Mahant S, Johnston BC. Probiotics for the prevention of pediatric antibiotic-associated diarrhea. Cochrane Database of Systematic Reviews 2015, Issue 12. Art. No.: CD004827. DOI: 10.1002/14651858.CD004827.pub4
2. Friedrich Schiller University, Institute of Nutritional Science, Jena, German.Long-term consumption of fermented dairy products over 6 months increases HDL cholesterol. September 2002, Volume 56, Number 9, Pages 843-849
3. Broeckx, Ge ́raldine, Vandenheuvel, Dieter, Claes, Ingmar J.J., Lebeer, Sarah, Kiekens, Filip, Drying techniques of probiotic bacteria as an important step towards the development of novel pharmabiotics.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.04.002
4. Lievense, L.C., van‟t Riet, K., 1994. Convective drying of bacteria II. Factors influencing survival. Adv. Biochem. Eng. Biotechnol. 51, 71–89. doi:10.1007/BFb0008734
5. Ghandi, A., Powell, I.B., Howes, T., Chen, X.D., Adhikari, B., 2012. Effect of shear rate and oxygen stresses on the survival of Lactococcus lactis during the atomization and drying stages of spray drying: A laboratory and pilot scale study. J. Food Eng. 113, 194–200. doi:10.1016/j.jfoodeng.2012.06.005
6. Behboudi-Jobbehdar, S., Soukoulis, C., Yonekura, L., Fisk, I., 2013. Optimization of spray- drying process conditions for the production of maximally viable microencapsulated L. acidophilus NCIMB 701748. Dry. Technol. 31, 1274–1283. doi:10.1080/07373937.2013.788509
7. Poolman, B., 2002. Transporters and their roles in LAB cell physiology. Antonie van Leeuwenhoek, Int. J. Gen. Mol. Microbiol. 82, 147–164. doi:10.1023/A:1020658831293
8. Santivarangkna, C., Kulozik, U., Foerst, P., 2008b. Inactivation mechanisms of lactic acid starter cultures preserved by drying processes. J. Appl. Microbiol. 105, 1–13. doi:10.1111/j.1365-2672.2008.03744.x
9. Garvey, C.J., Lenné, T., Koster, K.L., Kent, B., Bryant, G., 2013. Phospholipid membrane protection by sugar molecules during dehydration-insights into molecular mechanisms using scattering techniques. Int. J. Mol. Sci. 14, 8148–8163. doi:10.3390/ijms14048148
10. Bielecka, M., Majkowska, A., 2000. Effect of spray drying temperature of yoghurt on the survival of starter cultures, moisture content and sensoric properties of yoghurt powder. Nahrung 44, 257–260. doi:0027-769X/2000/0407-0257S17.50+.50/0
11. Lebeer, S., Vanderleyden, J., De Keersmaecker, S.C.J., 2008. Genes and molecules of lactobacilli supporting probiotic action. Microbiol. Mol. Biol. Rev. 72, 728–764. doi:10.1128/MMBR.00017-08
12. Jalali, M., Abedi, D., Varshosaz, J., Najjarzadeh, M., Mirlohi, M., Tavakoli, N., 2012. Stability evaluation of freeze-dried Lactobacillus tolerance and Lactobacillus delbrueckii subsp. bulgaricus in oral capsules. Res. Pharm. Sci. 7, 31–36.
13. Jofré, A., Aymerich, T., Garriga, M., 2015. Impact of different cryoprotectants on the survival of freeze-dried Lactobacillus rhamnosus and Lactobacillus casei/paracasei during long- term storage. Benef. Microbes 6, 381–386. doi:10.3920/BM2014.0038
14. Yamaguchi A, Tanaka S, Yamaguchi S, Kuwahara H, Takamura C, et al. (2012) Two Novel Heat-Soluble Protein Families Abundantly Expressed in an Anhydrobiotic Tardigrade. PLoS ONE 7(8): e44209. doi:10.1371/journal.pone.0044209
15. Tunnacliffe A, Lapinski J, McGee B. (2005) A putative LEA protein, but no trehalose, is present in anhy- drobiotic bdelloid rotifers. Hydrobiologia 546: 315–321.
16. Hengherr S, Heyer AG, Kohler H-R, Schill RO. (2008) Trehalose and anhydrobiosis in tardigrades-evi- dence for divergence in responses to dehydration. FEBS J. 275: 281–288. PMID: 18070104
17. Zhang, Z.-Q. (2011). "Animal biodiversity: An introduction to higher-level classification and taxonomic richness" . Zootaxa. 3148: 7–12.
18. Rebecchi, L.; et al. "Two Tardigrade Species On Board the STS-134 Space Flight" in "International Symposium on Tardigrada, 23–26 July 2012" . p. 89. Retrieved 2013-01-14.