Team:Stanford-Brown/Biomining/Harvesting

Harvesting

The FliC gene codes for flagellin, a protein that arranges itself in a hollow cylinder to form the filament of a bacterial flagellum. The N and C termini of flagellin and alpha-helices flanking the termini form the inner core of the filament cylinder (Bergman). The central portion, or “dispensable region,” forms the outer surface of the filament, and is highly variable.

iGEM Team Slovenia 2008 used the FliC gene to design a chimeric fusion protein expressing the antigenic segment of H. pylori on E. coli flagella (BBa_K133038). Our team wanted to use the same flagellar expression mechanism to express metal binding sequences, and our part is an improvement on the Slovenian biobrick. In the process, we designed the FliC gene with a multiple cloning site (MCS) in the dispensable region so that any iGEM team can insert a protein to be expressed in the dispensable region.

Engineered MCS FliC:

Biobricks used: Promoter J23100, RBS B0030, Terminator B0015

We inserted the following metal-binding sequences into the FliC disposable region:

"HTTC" (Cu+2)

HNLGMNHDLQGERPYVTEGC -->CATAACCTGGGCATGAACCATGATCTGCAGGGCGAACGCCCGTATGTGACCGAAGGCTGC

"HypB1" (Cu+1)

CTTCGCG -->TGCACCACCTGCGGCTGCGGC

"HypB2" (Cu+1)

MCTTCGCGEG -->ATGTGCACCACCTGCGGCTGCGGCGAAGGC

HTTC4 - inserted fully

HTTC6 - close to perfect insertion (couple of bases off)

HTTC8 - inserted fully

HTTC9 - inserted fully

HTTC10 - close to perfect insertion

HypB14 - inserted fully

HypB24 - inserted fully

HypB24 - inserted fully

Undergoing metal-binding assays now and flagella imaging on SEM.

Flagella Removal Protocol

Procedure from Westerlund-Wikstrom Paper Cited

For large scale removal and purification of the flagella, we hoped to adopt the purification method stated in the paper entitled “Functional expression of adhesive peptides on flagellin”. The method outlined involved four steps:

1. Shearing of flagella from bacteria

2. Separating the cells from flagella

3. Purifying the flagella

4. Detection of the flagella

In the first step, agar plates of the bacteria were collected in Tris-HCL buffer. The cells then had their flagella sheared off by a Turrax homogenizer at 20,000 r.p.m. The next step included pelleting the cells, and re-centrifuging the supernatant to ensure that all residual bacterium were removed. The supernatant, which contained the flagella, was then ultracentrifuged to form a pellet which was reconstituted in 1ml 10mM Tris-HCL and 0.5% deoxycholate. The third step then involved purifying the flagella. This was accomplished by isopycnic ultracentrifugation in a 10-60% sucrose gradient in Tris-DOC. The complete conditions for ultracentrifugation are outlined within the article. Concentrated light was then used to illuminate and detect the flagella, which were then collected and dialysed against Tris and distilled water.

Once the flagella have been collected, we would need to separate the metal ions for collection and use. The purified intact flagella (complete with metal binding peptides and metal ions) would be collected on Gelman cellulose acetate filters. The Tris-DOC buffer would be washed through with 0.5M KCL. The filter and flagella were then immersed in 5M urea - 0.05M KCL for half an hour at 26 C. At this point, the filaments (flagellin) dissociated while the hook-basal body complexes that make up the rest of the flagella were eluted.

CITATIONS

Molly A. Bergman,Lisa A. Cummings,Robert C. Alaniz,Laura Mayeda,Ivana Fellnerova,Brad T. Cookson. CD4+-T-Cell Responses Generated during Murine Salmonella enterica Serovar Typhimurium Infection Are Directed towards Multiple Epitopes within the Natural Antigen FliC. 2005 Infect Immun. 73(11): 7226–7235

E.Coli FliC gene: http://biocyc.org/ECOLI/sequence-rc?type=GENE&object=EG10321

Kouichi Kuroda and Mitsuyoshi Ueda. Molecular design of the microbial cell surface toward the recovery of metal ions. Current Opinion in Biotechnology 2011, 22:427–433

Benita Westerlund-Wikstro ̈m, Jarna Tanskanen, Ritva Virkola, Jo ̈rg Hacker, Martin Lindberg, Mikael Skurnik and Timo K.Korhonen. Functional expression of adhesive peptides on flagellin (Section - Purification of Chimeric Flagella) Protein Engineering vol.10 no.11 pp.1319-1326, 1997