production of turnip yellow mosaic virus nano-containers from lactococcus lactis for zinc...
TRANSCRIPT
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Production of Turnip yellow mosaic virus nano-containers
from Lactococcus lactis for zinc fortification
Alma Laney
Dr. Theo Dreher Lab
Department of Microbiology
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Nutrition in America
• Many Americans, 10%, have a deficiency in zinc levels, somewhat due to the negative interactions with grains and beans which causes the zinc to become unavailable for use.
• Zinc is part of the active site for many enzymes.
• Zinc is especially essential for pregnant and nursing mothers.
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TYMV capsids for micronutrient delivery
• Lactococcus can be modified to grow cheese and yogurt that is zinc enriched and help solve the problem of zinc deficiency.
• The problem is how to protect the zinc from chelation by phytates during digestion.
• What if the zinc was protected by a shell that would eventually break down later in digestion process?
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Turnip yellow mosaic virus could solve this problem
• The outer and inner surface of TYMV capsids contain several sites for modification by chemical reactions.
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Turnip yellow mosaic virus general background
• The TYMV capsid is made of 180 copies of the same protein monomer.
• The capsid icosahedral and 28 nm in diameter
• Infects crucifers
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What is a minimal zinc finger?
• A minimal zinc finger is the minimal number (25) of amino acids required to effectively bind zinc and be functional.
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Project overview
• Production and purification of TYMV capsids in E. coli• Production in Lactococcus• Creating a minimal zinc finger within the TYMV capsid.• Future work
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Predictions
• The TYMV empty capsids can be formed in E. coli and be purified.
• The TYMV empty capsids can also be produced by Lactococcus.
• The zinc finger will not interfere with capsid assembly.
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Production and purification in E. coli
• The monomer has been expressed in E. coli BL21 previously.
• The empty capsid was also produced and purified from E. coli pLysS.
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Production in E. coli
TYMV CP [570bp]
Nde I [Start of transcription] Hind IIIStop of transcription [100 bp] upstream from Hind III
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Production cont.• The capsid was induced using 1mM IPTG
at 28 degrees C for 9 or more hours.
The cells were then lysed using lysozyme and sonication. The capsids were then purified by size exclusion using a Centricon.
Lac promoterTYMC gene
IPTG
RNA Pol
Repressor
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Purification in E. coli
Lane 1: Protein ladderLane 2: Pre-inducedLane 3: Post-inducedLane 4: Insoluble fractionLane 5: Soluble fraction
Lane 1 2 3 4 5
120 kDa
20 kDa
26 kDa34 kDa47 kDa86 kDa
21 kDa
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Production in Lactococcus
• The TYMC gene was amplified using PCR.
• The PCR primers contained restriction sites to expedite ligation into the shuttle vector.
Pst I
Xba ITYMV CP
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The shuttle vector
• The shuttle vector is pBG568 pipwt.
Pip wt gene
Xba I
Xho I
Pst I
OriC
Ori [Lactococcus]
Erythromycin
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Continuing work in Lactococcus
• The TYMV capsid gene will then be ligated into pBG568, a shuttle vector that contains the ori for both E. coli and Lactococcus.
• The sequence will then be verified by restriction digest and sequencing the gene of interest.
• The Lactococcus will then be induced to produce the empty TYMV capsid.
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Continuing work in Lactococcus
• The capsid will then be purified and verified as correct by electron microscopy and by SDS-PAGE gel electrophoresis.
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Continuing work in E. coli
• The capsid gene will then be altered with a minimal zinc finger inside the formed capsid.
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Zinc finger addition
• The zinc finger was generated using mega-primers that include the start of the TYMV CP
• The product then had 20 cycles of PCR to amplify the sequence and to place restriction sites for insertion into the TYMV CP.
Minimal zinc finger sequence + start of TYMV CP
Nde IPvu II
TYMV CPNde I Pvu II
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Zinc finger cont.
Zinc finger insert [~190bp]
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Continuing work
• The empty zinc modified capsid will then be purified and checked for zinc content.
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Future work• Produce empty capsids
of different sizes.• Produce cheese that
has been zinc enriched.• Test to see if the
TYMV-zinc capsids will survive the stomach and be digestible in the small intestine.
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Acknowledgements
• The Howard Hughes Medical Institute
• Dr. Theo Dreher
• Dr. Bruce Geller
• Josh Powell
• Dr. Yannis Tzanetakis
• The Theo Dreher Lab