Confirmation of positive
clones
Screening of positive
clones
Selection of high copy
number clones
Selection of positive clones
FLUTCORE vaccine yeast constructs
Construct design
Bacterial cloning
Molecular and morphological
Characterization
Yeast cloning
Cell bank
Molecular cloning
Constructs design
Synthesis of inserts (GeneArt )
Standard cloning manipulation
(restriction digestion and ligation)
Heterotandem construct PHe7K1K1
Bacterial cloning
Cloning of inserts into pPICZ C plasmid using specific restriction
sites
Plasmid transfection into E. Coli DH5α using the heat-shock
method
Growth of positive clones using selective medium (low salt LB agar
containing 0.1 mg/ml of zeocin)
pPICZ C plasmid
Screening and confirmation of positive clones
Screening of positive clones via colony PCR using insert specific primers
M 1 2 3 4 5 6
M: DNA ladder1: Clone 12: Clone 23: Clone 34: Clone 45: Clone 56: Negative control
Sequencing of inserted DNA fragments of two positive clones
Yeast cloning
Plasmid linearization using pmeI restriction site (present on the plasmid sequence)
P. Pastoris KM71H transformation (electroporation) using linearized plasmids (5 µl) and electro competent yeast cells (80 µl)
Incubation of transformed cells in:1 ml of 1 M sorbitol at 30° C for 90 minutes and
10 ml of YPD at 30° C and 250 rpm for 90 minutes
Note: no antibiotics are used at tis stage
Yeast cloning
More inserts
=More antibiotic resistance gene
Integration of zeocin resistance gene together with target inserts
Selection of high copy clones: zeocin
Selection of 24 clones (pool of cells) showing the highest OD600
gDNA extraction of the selected clones
Growth of cloned yeast cells using selective medium: YPDS with increasing concentrations of zeocin (0.2 and 2 mg/ml)
Selection of high copy clones: qRT-PCR
Selection of the highest copy clone (pool of cells) via qPCR
Single-cell colonies
Isolation of 8 single-cell colonies on YPD agar
Growth of selected single-cell colonies and gDNA extraction
Selection of the highest copy clone via qPCR
Yeast Clones
Clone Core 1 Core 2Product MW
(KDa)
1 K1 K1 42.5
2 LAH HA2.3 3sp-(M2)3-3sp 58.8
3 LAH H3 K1 48.2
4 3sp-LAH H1/H3/HB-3sp 3sp-(M2)3-3sp 71.6
5 LAH H1/H3 3sp-(M2)3-3sp 63.7
6 LAH H3 3sp-(M2)3-3sp 56.8
7 K1 LAH H3 48.2
8 M2a-LAH H1-M2b LAH H3 58.9
9 LAH H1 3sp-(M2)3-3sp 56.8
10 LAH H1 LAH H3 53.7
11 M2a-LAH H1-M2b M2a-LAH H3-M2c 64.1
12 K1 3sp-(M2)3-3sp 51.2
VLP1
VLP2
Research Cell Bank (RCB)
Master Cell Bank (MCB) and Working Cell Bank (WCB) generated and analysed following the same criteria used for the RCB
RCB
Morphology
Cell viability
Insert copy number
Genetic identity
Stocks of P. pastoris KM71H transformed with VLP coding sequences
RCB generation and characterization
Single-cell colony stocks
VLP1 VLP2
Samples inoculated onto YPD agar (with no antibiotic) and incubated at 30° C for 72 hours
VLP1 morphology
Liquid culture 1 day at -80° C
6 days at -80° C 30 days at -80° C
Samples inoculated onto YPD agar (with no antibiotic) and incubated at 30° C for 72 hours
VLP2 morphology
Liquid culture 1 day at -80° C
6 days at -80° C 30 days at -80° C
Samples inoculated onto YPD agar (with no antibiotic) and incubated at 30° C for 72 hours
Morphology
Colour Form Margin Elevation Colony size (mm)
Liquid culture White Rounded Entire Raised 2.87±0.13
Stocks after 1 day at - 80°C White Rounded Entire Raised 2.08±0.10
Stocks after 6 days at - 80°C White Rounded Entire Raised 2.23±0.08
Stocks after 30 days at -
80°CWhite Rounded Entire Raised 2.6±0.06
Colour Form Margin Elevation Colony size (mm)
Liquid culture White Rounded Entire Raised 2.64±0.11
Stocks after 1 day at - 80° C White Rounded Entire Raised 2.85±0.08
Stocks after 6 days at - 80°
CWhite Rounded Entire Raised 2.71±0.14
Stocks after 30 days at -
80° CWhite Rounded Entire Raised 2.82±0.04
VLP2
Cell viability: CFU determination
RCB stock before freez-
ing
RCB stock after 1 day at -80° C
RCB stock after 6 days
at -80° C
RCB stock after 30 days
at -80° C
0.0E+00
5.0E+07
1.0E+08
1.5E+08
2.0E+08
CF
U/m
L
VLP1 VLP2
0.00E+00
5.00E+07
1.00E+08
1.50E+08
2.00E+08
CF
U/m
L
Serial dilution (1:10) of stock samples were prepared, inoculated onto YPD agar (with no antibiotic) and incubated at 30° C for 72 hours
Insert copy number: qRT-PCR
RCB stock before freezing
RCB stock after one day at -80° C
RCB stock after six days at -80° C
0
10
20
30
40
50
60
Gen
e co
py n
umbe
r
VLP1 VLP2
RCB stock before freezing
RCB stock after one day at -80° C
RCB stock after six days at -80° C
0
10
20
30
40
50
60
Gen
e co
py n
umbe
r
gDNA extracted from each sample was used to perform qRT-PCR
Genetic identity: PCR
Amplification of the entire VLP coding fragments
M: DNA ladder1: VLP1 single-cell stock2: VLP1 RCB stock before storage3: VLP1 RCB stock after storage4: VLP1 negative control5: VLP2 single-cell stock6: VLP2 RCB stock before storage7: VLP2 RCB stock after storage8: VLP2 negative control
Genetic identity: sequencing
Full VLP sequence
Pic upstream F
Mid core1 F
SacI F
SalI R
NheI R
Pic downstream R
Correspondence between expected and cloned nucleotide sequences
Conclusions
1. Cloning of VLP coding sequences into P. pastoris KM71H
2. Isolation of single-cell clones
3. Selection of the highest copy number clones
4. Generation and characterization of cell banks
a. Same cell morphology before and after stock storage at -80° C
b. No significant differences in cell viability before and after storage
at -80° C and at different time points
c. Stability of insert copy number before and after storage at -80° C
d. Proof of genetic identity
P. Pastoris KM71H