mercury resistance and accumulation in escherichia coli with cell surface expression of fish...
TRANSCRIPT
Mercury resistance and accumulation in
Escherichia coli with cell surface expression of
fish metallothionein
Kristen Soroka
http://buyorganictilapia.com/
Kuo-Hsing et al. 2010
How does Mercury get into the environment?
http://www.nap.edu/openbook.php?isbn=0309071402&page=17
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
What is Metallothionein (MT)?
Capasso et al. 2003
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
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>.
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
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).
Primers
MT Expression in Cytosol
• Kanamycin resistance
• IPTG
• SDS-14% PAGE gel
Kuo-Hsing et al. 2010, Fig 1a.
No MT Hmt Mmt Tmt
Cellular Location of Cytocolic tMT
Kuo-Hsing et al. 2010, Fig 2b.
Alexa Flour 594 donkey anti-rabbit IgG antibody
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
Cellular Labeling of outer membrane targeted tMT
Kuo-Hsing et al. 2010, Fig 2a.
Alexa Flour 488 donkey anti-rabbit IgG antibody
Disk Assay and Resistance
Kuo-Hsing et al. 2010
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
Kuo-Hsing et al. 2010
Cytosol expression
Outer membrane expression
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)
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)
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
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>.