mercury resistance and accumulation in escherichia coli with cell surface expression of fish...
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Mercury resistance and accumulation in
Escherichia coli with cell surface expression of
fish metallothionein
Kristen Soroka
http://buyorganictilapia.com/
Kuo-Hsing et al. 2010
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How does Mercury get into the environment?
http://www.nap.edu/openbook.php?isbn=0309071402&page=17
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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
• Headaches
• Tremors
• Emotional changes
• Neuromuscular changes
• Kidney effects
• Respiratory failure
• Death
http://www.epa.gov/hg/effects.htm
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What is Metallothionein (MT)?
Capasso et al. 2003
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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
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pET-30aCytosol expressionHuman (mt1A) = pET-HmtMouse (mt1) = pET-MmtTilapia (mt) = pET-Tmt
Cell Surface ExpressionControl = pET-OmpCHuman = pET-OmHmtMouse = pET-OmMmtTilapia = pET-OmTmt
<http://www.synthesisgene.com/vector/pET-30a.pdf>.
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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
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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).
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Primers
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MT Expression in Cytosol
• Kanamycin resistance
• IPTG
• SDS-14% PAGE gel
Kuo-Hsing et al. 2010, Fig 1a.
No MT Hmt Mmt Tmt
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Cellular Location of Cytocolic tMT
Kuo-Hsing et al. 2010, Fig 2b.
Alexa Flour 594 donkey anti-rabbit IgG antibody
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MT Expression in Outer Membrane
• Similar expression methods
• Isolating the nonpolar membrane protein.
• SDS-14% PAGE gel
Kuo-Hsing et al. 2010, Fig 1b.
OmTm
tOm
Hmt
OmM
mt
pET-30a
pET-Om
pC
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Cellular Labeling of outer membrane targeted tMT
Kuo-Hsing et al. 2010, Fig 2a.
Alexa Flour 488 donkey anti-rabbit IgG antibody
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Disk Assay and Resistance
Kuo-Hsing et al. 2010
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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
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Kuo-Hsing et al. 2010
Cytosol expression
Outer membrane expression
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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)
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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)
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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
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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 <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>.