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Kristen Soroka

Kuo-Hsing et al. 2010. Mercury resistance and accumulation in Escherichia coli with cell surface expression of fish metallothionein. http:// buyorganictilapia.com /. Kristen Soroka. How does Mercury get into the environment?. http:// www.nap.edu/openbook.php?isbn =0309071402&page=17.

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Kristen Soroka

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

  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 Elemental Mercury Headaches Tremors Emotional changes Neuromuscular changes Kidney effects Respiratory failure Death • Attacks the nervous system of humans and developing fetuses • Vision, speech, hearing, or walking impairment • Disturbance in sensations • Muscle weakness 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 <http://www.synthesisgene.com/vector/pET-30a.pdf>.

  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 No MT Hmt Mmt Tmt • Kanamycin resistance • IPTG • SDS-14% PAGE gel Kuo-Hsing et al. 2010, Fig 1a.

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

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

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

  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 • Hocking River • Mill Creek • Clear Creek • Gold Shop Mercury Capture System http://www.epa.gov/international/toxics/mercury/asgm.html

  20. References • Capasso, Clemente, VincenzoCarginale, Orlando Crescenzi, Daniela Di Maro, ElioParisi, Roberta Spadaccini, and Piero Andrea Temussi. "Solution Structure of MT_nc, a Novel Metallothionein from the Antarctic Fish Nototheniacoriiceps." Cell Press 11 (2003): 435-43. ScienceDirect. 1 Dec. 2013 <http://www.sciencedirect.com/science/article/pii/S0969212603000443>. • 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. <http://link.springer.com/article/10.1007/s00253-010-2466-x>. • "Health Effects." EPA. 9 July 2013. Environmental Protection Agency. 01 Dec. 2013 <http://www.epa.gov/hg/effects.htm>. • "PET-30a-c(+) Vectors." Map. Novagen. Dec. 1998. 1 Dec. 2013 <http://www.synthesisgene.com/vector/pET-30a.pdf>. • "Reducing Mercury Pollution from Artisanal and Small-Scale Gold Mining." EPA. 29 Aug. 2013. Environmental Protection Agency. 03 Dec. 2013 <http://www.epa.gov/international/toxics/mercury/asgm.html>. • "Zone of Inhibition Test for Antimicrobial Activity." Antimicrobial Test Laboratories. 02 Dec. 2013 <http://www.antimicrobialtestlaboratories.com/Zone_of_Inhibition_Test_for_Antimicrobial_Activity.htm>.

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