Team:NCTU Formosa/Safty

Researcher Safety

2.Before the commencement of our project,our team instructors and advisers have guided us to go through every expiriment to make sure everybody is well trained and fit for this project. 3.Everybody has to sterilize his/her hands and all the equipment platforms with alcohol solution(70%) before and after each expiriment. 4.Emergency equipments are well prepared and all the researchers understand how to use these equipments and their exact position. 5.It is not allowed that researcher carries experiment alone.There must be at least two members to conduct the experiment. 6.Food and beverages are not allowed to appear in the lab </div></div>




Public safety

1.All collected lab waste are sterilized, packaged and executed. 2.E.coli used in our lab is less competitive than wild type, because they are unable to form biofilms and to thrive in the intestine. 3.Liquid waste that contains E.coli is disinfected by adding peroxides and cleaners before it is disposed.




Instruments in our lab

1.incubator The orbital shaker incubator is widely used in microbiology labs, cell biology labs, etc. It provides a stationary temperature to cultivate bacteria and cells. Some incubator also have functions to maintain the humility level. In our lab, the incubator is maintaining the temperature at 37 degrees Celsius, which is the optical temperature of E.coli growing. In this year, our team was authorized to work with an non-pathogenic bacterial strain, the Escherichia coli DH5. The strain ais regularly employed in research, industry and study. Biosafety Level 1 standard regulations were strictly followed while using these strains.







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Figure 2. Phycocyanobilin is the part of photoreceptor that responds to light, and it is not naturally produced in E.coli,so we introduced two phycocyanobilin-biosynthesis genes (ho1 and pcya) from Synechocystis that convert heme into phycocyanobilin.

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Figure 3. The light receptor cph8 is composed of cph1(pink) and envZ-ompR(maroon).




Design of Red promoter

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Reference

1. part BBa_I15008;MIT Registry of Standard Biological Parts
2. part BBa_I15009;MIT Registry of Standard Biological Parts
3. Levskaya, A. et al .(2005). Engineering Escherichia coli to see light. Nature, 438(7067), 442.
4. Kehoe DM, Grossman AR (1996) Similarity of a chromatic adaptation sensor to phytochrome and ethylene receptors. Science 273(5280):1409–1412
5. Yeh KC, Wu SH, Murphy JT, Lagarias JC (1997) A cyanobacterial phytochrome two-component light sensory system. cience 277 (5331):1505–1508
6. Dutta R, Qin L, Inouye M (1999) Histidine kinases: diversity of domain organization. Mol Microbiol 34(4):633–640
7. Forst SA, Roberts DL (1994) Signal transduction by the EnvZ–OmpR phosphotransfer system in bacteria. Res Microbiol 45(5–6):363–373
8. Thomas Drepper, Ulrich Krauss,Sonja Meyer zu Berstenhorst, Jörg Pietruszka, Karl-Erich Jaeger.(2011).Lights on and action! Controlling microbial gene expression by light. Appl Microbiol Biotechnol, 90:23–40 DOI:10.1007/s00253-011-3141-6

Biobrick Design





Figure.1





Figure.2

We searched the DNA sequence of the PBANs from moths on NCBI, then contrasted to the amino sequences from papers so that we can selected the DNA fragments which directly correspond to gland-stimulating function, By ligating Ribosome binding site(B0034) and PBAN DNA sequence, we were able to make E.coli directly produce these PBANs instead of the original complex process of PBAN biosynthesis in insects. We had gotten nine kinds of PBANs,each of which is from one kind of moth ,after we constructed the PBAN biobricks, the B0034+BFP+J61048 biobrick was ligated behind the PBAN biobrick in order to model each PBAN biobrick respectively in the future and make observing the production of PBANs more convenient for us.










Reference

9. Torsten Waldminghaus, Nadja Heidrich, Sabine Brantl and Franz Narberhaus .(2007). FourU: a novel type of RNA thermometer in Salmonella . Molecular Microbiology , 65(2): 413–424 DOI:10.1111/j.1365-2958.2007.05794.x
10. part BBa_K115002;TUDelft Registry of Standard Biological Parts

Device

Introduction

Mechanism

Design

Reference

11. Xu, S.; Montgomery, M.; Kostas, S.; Driver, S.; Mello, C. (1998). "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans". Nature 391 (6669): 806–811 DOI:10.1038/35888
12. Jörg Vogel , Ben F. Luisi.(2011). Hfq and its constellation of RNA. Nature Reviews Microbiology, 9:578-589
13. E.K. Jocelyn, S.G. Elliott , T.K. Stephen, "Lewin's Genes X.-10th ed.", Jones & Bartlett, Sudbury, MA, 2011.
14. Karen M. Wassarman.(2002). "Small RNAs in Bacteria: Diverse Regulators of Gene Expression in Response to Environmental Changes". Cell, 109:141–144
15. Hongmarn Park, Geunu Bak, Sun Chang Kim & Younghoon Lee.(2013). "Exploring sRNA-mediated gene silencing mechanisms using artificial small RNAs derived from a natural RNA scaffold in Escherichia coli ". Nucleic Acids Research,Vol. 41, No. 6, 3787-3804 DOI:10.1093
16. Vandana Sharma, Asami Yamamura & Yohei Yokobayashi.(2011). "Engineering Artificial Small RNAs for Conditional Gene Silencing in E. coli". ACS Synthetic Biology




Cover image credit: DVQ