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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 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. 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? Turnip yellow mosaic virus could solve this problem • The outer and inner surface of TYMV capsids contain several sites for modification by chemical reactions. 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 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. 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 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. 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. Production in E. coli Nde I [Start of transcription] Stop of transcription [100 bp] upstream from Hind III TYMV CP [570bp] Hind III Production cont. • The capsid was induced using 1mM IPTG at 28 degrees C for 9 or more hours. IPTG RNA Pol Repressor Lac promoter TYMC gene The cells were then lysed using lysozyme and sonication. The capsids were then purified by size exclusion using a Centricon. Purification in E. coli Lane 1: Protein ladder Lane 2: Pre-induced Lane 3: Post-induced Lane 4: Insoluble fraction Lane 5: Soluble fraction Lane 1 2 120 kDa 86 kDa 47 kDa 34 kDa 26 kDa 21 kDa 20 kDa 3 4 5 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 TYMV CP Xba I The shuttle vector • The shuttle vector is pBG568 pipwt. Pst I OriC Xho I Pip wt gene Xba I Ori [Lactococcus] Erythromycin 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. Continuing work in Lactococcus • The capsid will then be purified and verified as correct by electron microscopy and by SDS-PAGE gel electrophoresis. Continuing work in E. coli • The capsid gene will then be altered with a minimal zinc finger inside the formed capsid. Zinc finger addition • The zinc finger was generated using megaprimers 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 Pvu II Nde I Nde I Pvu II TYMV CP Zinc finger cont. Zinc finger insert [~190bp] Continuing work • The empty zinc modified capsid will then be purified and checked for zinc content. 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. Acknowledgements • • • • • • The Howard Hughes Medical Institute Dr. Theo Dreher Dr. Bruce Geller Josh Powell Dr. Yannis Tzanetakis The Theo Dreher Lab