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