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 TYMV CP [570bp] Nde I [Start of transcription] Hind III Stop of transcription [100 bp] upstream from Hind III

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 promoter TYMC gene IPTG RNA Pol Repressor

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 kDa 20 kDa 26 kDa 34 kDa 47 kDa 86 kDa 21 kDa

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 I TYMV CP

The shuttle vector The shuttle vector is pBG568 pipwt. Pip wt gene Xba I Xho I Pst I OriC 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 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 I Pvu II TYMV CPNde IPvu II

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