Vicugna pacos
isolate
'Prince'
collection date
2020-11-04
geo_loc_name
USA: New Haven
isolation source
phage display library
synthetic
True
sex
male
age
not collected
dev_stage
adult
age_event
sampling
population
not applicable
ethnicity
not applicable
race
not applicable
strain
not applicable
study_group_description
small-scale cell-based phage display panning
disease
not applicable
disease_length
not applicable
disease_stage
not applicable
prior_therapies
not applicable
treatment
not applicable
medical_history
not applicable
tissue
peripheral blood lymphocytes
anatomic_site
peripheral blood
health_state
not applicable
collection_time_point_reference
panning_start
collection_time_point_relative
7
collection_time_point_relative_unit
id: UO:0000033, label: day
biomaterial_provider
Barbara Kazmierczak, Yale University, Ste S140, 300 Cedar Street, New Haven, CT 06519
tissue_processing
phage elution by hydrochloric acid/triethylamine elution
cell_type
phage display
cell_phenotype
not applicable
single_cell
False
cell_number
not collected
cells_per_reaction
not collected
cell_storage
Y
cell_quality
not collected
cell_isolation
not applicable
target_antigen
pilus
phage_display_bait_type
bacterial cells
selection_name
024e.3.1
counter_selection_cell_genotype
∆pilA
counter_selection_cell_background
PA103
counter_selection_cell_strain_number
213.0
counter_selection_cell_growth_condition
exponential phase
counter_selection_cell_growth_protocol
a single colony was used to inoculate a culture of LB which was grown shaking at 37 °C overnight, then subcultured 1/25 into the same media and grown for 3 hours.
selection_cell_genotype
WT
selection_cell_background
PA103
selection_cell_strain_number
209.0
selection_cell_growth_condition
exponential phase
selection_cell_growth_protocol
a single colony was used to inoculate a culture of LB which was grown shaking at 37 °C overnight, then subcultured 1/25 into the same media and grown for 3 hours.
technical_replicate
1
cell_processing_protocol
Library of phage-displayed VHHs were applied to counter-selection bacterial cells pre-washed with PBS+Ca+Mg and incubated 1 hour; bacteria removed by centrifugation 3750 rcf x 5 min, then supernatant applied to pre-washed selection bacterial cells and incubated 2 hours. Bacteria harvested by centrifugation, washed 4 times with PBS + Ca + Mg. Phage were eluted from bacterial cells by addition of 0.1 N HCl x 5 min, neutralization with 1 M Tris-HCl pH 11; bacteria harvested and second elution performed with 0.1 M triethylamine (TEA) x 5 min and neutralization with Tris pH 6.8. Acid- and base- eluates combined. 10 µL of eluate used as template.
immunogen
Four P. aeruginosa laboratory strains---PA14 (serotype O10), PAO1 (O5), PAK (O6) and PA103 (O11)---were grown to exponential phase in liquid LB and overnight on LB agar; cells were harvested, lysed by french press (>14,000 psi), and the lysate clarified by centrifugation. Membrane proteins were enriched by centrifugation through sucrose density gradients. Additionally, PA14 and PAO1 were grown as static biofilms in petri dishes in both LB and M9 minimal media at 30 ˚C for 36 hours, harvested by scraping, and lysed as above. The soluble proteins, enriched membrane proteins, and biofilm extracts were pooled to generate a mixed antigen preparation.
phage_display_selection_round
4
phage_display_selection_input_or_output
output
phage_display_selection_selection_or_counterselection
selection
BioSampleModel
Model organism or animal