1. Mercury resistance and accumulation in Escherichia coli with cell surface expression of fish metallothionein Kristen Soroka http://buyorganictilapia.com/ Kuo-Hsing et al. 2010

2. How does Mercury get into the environment? http://www.nap.edu/openbook.php?isbn=0309071402&page=17

3. What are the health effects of Mercury? Methyl Mercury Attacks the nervous system of humans and developing fetuses Vision, speech, hearing, or walking impairment Disturbance in sensations Muscle weakness Elemental Mercury http://www.epa.gov/hg/effects.htm

4. What is Metallothionein (MT)? Capasso et al. 2003

5. Eight Plasmids! A control plasmid for the cytosol expression and outer membrane expression. Cytosol and outer membrane expression for: Human MT Mouse MT Tilapia MT

6. pET-30a Cytosol expression Human (mt1A) = pET-Hmt Mouse (mt1) = pET-Mmt Tilapia (mt) = pET-Tmt Cell Surface Expression Control = pET-OmpC Human = pET-OmHmt Mouse = pET-OmMmt Tilapia = pET-OmTmt.

7. Cytosol Plasmid Construct Control : pET-30a pMAL-p2X-MT1A encodes human MT (Hmt) and pMBP-TEV-MT1 encodes mouse MT (Mmt) (Kao et al. 2006) as cited in Kuo-Hsing et al. 2010 Tilapia liver (Tmt) Primers Cut-Sites : BglII and SacI

8. Outer Membrane Plasmid Construct Control : pET-30a  pET-OmpC OmpC gene from E. coli K19 Primers : OmpC-F and OmpC-R Cut-Sites : NdeI and BamHI Cut-Site : PstI “These plasmids were designed to express outer membrane fusion protein in which the MT was integrated into the seventh outer membrane loop of the OmpC protein” (Kuo-Hsing et al. 2010).

9. Primers

10. MT Expression in Cytosol Kanamycin resistance IPTG SDS-14% PAGE gel Kuo-Hsing et al. 2010, Fig 1a. No MTHmtMmtTmt

11. Cellular Location of Cytocolic tMT Kuo-Hsing et al. 2010, Fig 2b. Alexa Flour 594 donkey anti-rabbit IgG antibody

12. MT Expression in Outer Membrane Similar expression methods Isolating the nonpolar membrane protein. SDS-14% PAGE gel Kuo-Hsing et al. 2010, Fig 1b. OmTmt OmHmt OmMmt pET-30a pET-OmpC

13. Cellular Labeling of outer membrane targeted tMT Kuo-Hsing et al. 2010, Fig 2a. Alexa Flour 488 donkey anti-rabbit IgG antibody

14. Disk Assay and Resistance Kuo-Hsing et al. 2010

15. Disk Assay If the bacteria is susceptible the to the metal then a zone of inhibition will surround the plate The smaller the zone of inhibition means it is more resistant to the metal http://www.antimicrobialtestlaboratories.com/Zone_of_Inhibition_Test_for_Antimicrobial_Activity.htm

16. Kuo-Hsing et al. 2010 Cytosol expression Outer membrane expression

17. Sequence, Structure, and Function CXCC (mammalian)  CXXXCC (fish) (Sucdiero et al. 2005) as cited in Kuo-Hsing et al 2010 A change in primary structure influences the tertiary structure of proteins (Capasso et al. 2003) Structure dictates Function (Capasso et al. 2003, Fig 2)

18. Discussion “Our results showed that the limitation of intracellular accumulation could be overcome by anchoring the metal-binding proteins directly to the cell membrane for improving adsorption (Fig 3) and reducing the toxicity (Table 2)” (Kuo-Hsing et al. 2010)

19. More about Mercury http://fishadvisoryonline.epa.gov/General.aspx http://fishadvisoryonline.epa.gov/General.aspx Hocking River Mill Creek Clear Creek Gold Shop Mercury Capture System http://www.epa.gov/international/toxics/mercury/a sgm.html

20. References Capasso, Clemente, Vincenzo Carginale, Orlando Crescenzi, Daniela Di Maro, Elio Parisi, Roberta Spadaccini, and Piero Andrea Temussi. "Solution Structure of MT_nc, a Novel Metallothionein from the Antarctic Fish Notothenia coriiceps." Cell Press 11 (2003): 435-43. ScienceDirect. 1 Dec. 2013. Kuo-Hsing, Lin, Chien Mei-Gang, Hsieh Ju-Liang, and Huang Chieh-Chen. "Mercury resistance and accumulation in Escherichia coli with cell surface expression of fish metallothionein." Applied Microbiology and Biotechnology 87 (2010): 561-69. SpringerLink. 01 June 2010. Springer.. "Health Effects." EPA. 9 July 2013. Environmental Protection Agency. 01 Dec. 2013. "PET-30a-c(+) Vectors." Map. Novagen. Dec. 1998. 1 Dec. 2013. "Reducing Mercury Pollution from Artisanal and Small-Scale Gold Mining." EPA. 29 Aug. 2013. Environmental Protection Agency. 03 Dec. 2013. "Zone of Inhibition Test for Antimicrobial Activity." Antimicrobial Test Laboratories. 02 Dec. 2013.