Team:USP-Brazil/LaBioTec

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LaBioTec - Lab Diary

20 May - 24 May
Preparation of Lysis buffer using LiOAc (200 mM).
YPD Media preparation and autoclaving.
Plate media and store remaining volume
1g of SDS (1%) was added to the lysis buffer
Brought a plaque with Pichia pastoris (GS115) recently plated.

27 May - 7 June
Extract the genomic DNA of Pichia pastoris
Obs: In some wells, the water volume was completely evaporated.
Gradient PCR using the F_Paox1_GIB and R_Paox1_Kozak_GIB primers for P_AOX1 amplification and determine optimal temperature and DNA template concentration

T_melting: 55ºC, 58ºC, 60ºC, 62ºC and 65ºC
gDNA = 303.32 ng (10X), 30.332 ng (100X) e 3.0332 ng (1000X)

Verify on an agarose Gel if the PCR was successful

10 June - 14 June
To the evaporation test we fixed the styrofoam box on shaker.
The evaporation test was needed after an observation about the shaker’s coolers localization. It was found out that the hot wind generated for temperature adjustment cause an strong temperature gradient in some shaker areas.
Draw growth curve

17 June - 21 June
Evaluate the behaviour of Pichia pastoris growth with crescent ethanol concentration on media by performing a Pichia pastoris Ethanol Survival test protocol.
Some samples were also grown on cultures plates to qualitatively evaluate their viability.

1 July - 7 July
Evaluate the viability of P. pastoris through lyophilization process using a monosodium glutamate and milk as cryoprotectants
First, a sample of the inoculum was took and diluted in a range from 10^-1 to 10^-8 with YPD liquid media using serial dilution (180 uL of media, 20 uL of inoculum).
All dilutions were plated in triplicate and put to grow under 30 Celsius for 48 hours
4 different samples of the inoculum were put in 2,0 mL tubes and in small flasks, well mixed, fast frozen with liquid nitrogen, opened and put under lyophilization for 24 hours

	Inoculum	Milk	Glutamate	YPD media
1	-	-	-	500 uL
2	250 uL	-	-	250 uL
3	250 uL	250 uL	-	-
4	-	250 uL	-	250 uL

After lyophilization, the 4 different samples were suspended in 1 mL water and serially diluted from 10 to 10^-8 in YPD media, plated in triplicate and put to grow for 48 hours on 30 Celsius
Plates with the first inoculum and with the four different lyophilized samples were photographed and the colonies were counted.

10 July - 19 July
1 mL of inoculum was centrifuged and resuspended in 500 uL. The same were made with 2 mL of inoculum.
3 replicates of the first resuspended inoculum were mixed with 250 uL of milk solution and 250 uL of monosodium glutamate solution (1,2 and 3). The same was made with the second inoculum, but in only one sample.
All samples were lyophilized for 24 hours. A sample of 1 mL of the inoculum was plated under serial dilutions from 10^-1 to 10⁸.
The lyophilized samples were resuspended in 1 mL of water, serially diluted from 10^-1 to 10^-6 and plated.
The plates from the pure inoculum demonstrated growth after 24 hours and 30 Celsius, but not enough.

22 July - 10 August
Pichia were grown to the stationary phase and a new lyophilization was done. The initial OD was 0.314 for a 40 times dilution (OD of 12.56).
The lyophilization process went wrong ):
A new inoculum has been done and let to grow until the stationary phase.
Samples of inoculum were centrifuged and resuspended and mixed with milk solution and of monosodium glutamate solution as before
All samples but one were lyophilized for 24 hours. This one sample that was not lyophilized (control) was plated under serial dilutions from 10^-3 to 10^-8
To evaluate the viability of resuspended lyophilized samples in ethanol solutions 5 lyophilized samples were resuspended:

Two of them in 1 mL of water,
One in 10% Ethanol solution,
One in 15% Ethanol solution,
One in 20% Ethanol solution,

They were then serially diluted from 10^-1 to 10^-8 and plated from 10^-3 to 10^-8. The dilutions were plated like the previous tests. One resuspension in water was plated right after the addition of water (as a control). All other resuspensions were plated 4 hours after the end of lyophilization process to check the viability of cells after a possible genetic response time of our future genetic construction.
The resuspensions on YPD plates do not showed satisfactory results because of bad milk solution pipetting due to high viscosity. It is better to use fresh milk for lyophilization preparation Started the lyophilization process again with a new milk solution.

16 August - 20 August
Master Mix preparation
Precipitate 300uL of the digested material by ethanol
Transformation of competent cells with linearized pPIC9K-RFP with BgLII and purified by precipitation in ethanol by electroporation
Incubation on ice for 10min
Plaque in YPD/ gen 0,25mg/uL, YPD and MD

3 September
Repicking of colonies from plates Genet + YPD (3 colonies) and ND (14 colonies) in new plates of Genet + YPD.
Growth of the 7 candidates in medium with YPD + Genet + Methanol
The seven candidates mentioned above were tested in medium YPD + Genet + methanol to see with the RFP is expressed.
The suspensions made above were used to inoculate medium with geneticin and methanol. The concentrations used were:

Geneticin - 40uL in final volume of 10mL [4uL/mL]
Methanol - 50uL in final volume pf 10ml [5uL/mL] - 0,5%

gDNA extraction was performed

10 September - 11 September
Plates with MD and colonies - colony PCR

96 well plate:

1 Negative control
2 Non-transformed Pichia
3 Candidate
4 Candidate
5 Candidate
6 Candidate
7 Candidate
8 Candidate
9 Pichia - cultivation from MD plate
10 Pichia YPDgen 0,25 mg/mL
11 Pichia YPDgen 0,25 mg/mL
12 Duplicate from 9 (Pichia - cultivation from MD plate)

A 0
B 0,0078125
C 0,015625
D 0,03125
E 0,0625
F 0,125
G 0,25 mg/ml geneticin
H 1% Methanol/YPD 2uL

Resuspend colonies in YPD Inoculate
Plate YPDs/amtibiotic straight in YPD
PCR reaction and agarose gel
PCR reactions of the colonies A2,A5,A6,A8,B1,C1,C2,D1,D2,E1,F2 and G1 were made.
The reactions aim in the confirmation of the transformation from Pichia pastoris, using primers that identify the RFP sequence.
Colony D2 was plated in YPD 0,25 mg/mL and colonies formed used for fluorescence test.
To confirm that non transformed colonies didn´t amplified in our reaction we performed another reaction with non transformants, some other candidates from our plate and a previous positive test.
Colonies 2,5,6,7,9 and 12 showed growth. They were tested in regard to DO, except 12 which was a duplicate from 9.
Apparently all were sensible to antibiotics, except for 9.
Cultivation was homogenized and incubated again at 30°C/200rpm.

Cytometer:

20uL of cells grown for 24h in YPD/1% Methanol received 280uL of phosphate buffer 100mmM pHG.
Volume was transferred to cytometry tubes and analysed.

Apparently there were no induced cells, only on sample 12 there was 1,5% more cells with fluorescence.
Tests:

H2 - Control, non transformed and induced cell
H5,H6,H7,H9,H12 and X - candidates, transformed cells, grown in plates.

Obs: Cell X came from plate C, inducted with 0,5% methanol and 1% methanol.

18 September - 20 September
To check RFP expression a fluorescence plate test was performed

Readings every hour
Incubation: 30°C
Agitation: 0
Conditions tested: Medium FM22 - 50000 cels / well

M/E - Methanol/Ethanol mixture:

200 mM/200mM
200 mM/100mM
200 mM/12,5mM
100 mM/200mM
100 mM/100mM
100 mM/12,5mM
12,5 mM/200mM
12,5 mM/100mM
12,5 mM/12,5mM

Methanol:

400mM
200mM
100mM
50mM
25mM
12,5mM

Ethanol:

400mM
200mM
100mM
50mM
25mM
12,5mM

The computer restarted during the experiment, no results for this plate.
Fluorescence Plate test was redone (mixtures given in Methanol/Ethanol below)

1

2

3

4

5

6

7

8

9

10

11

12

A

fm22+cel

B

200/ 200

200/ 100

200/ 12,5

100/ 200

C

100/ 100

100/ 12,5

12,5/200

12,5/100

D

12,5/12,5

Met 400

Met 200

Met 100

E

Met 50

Met 25

Met 12,5

Met 6,25

F

Et 400

Et 200

Et 100

Et 50

G

Et 25

Et 12,5

fm22 w/o cel

H

fm22 w/o cel

Evaporation loss was intense. Video from results.

24 September
Fluorescence Plate test

Reading: every half hour
Incubation: 30°C
Agitation: 1mm
Agitation pattern: Orbital
Time under agitation: 13 min ON, 17 min OFF
Ex: 584 nm
Em: 619 nm
Gain: 63
Number of reads: 10
Integration time: 40 ms

Conditions tested:

YP (Yeast extract and Peptone)
FM22 (Basal salt medium)
YP + Sortbitol 20mM
Initial OD (Optic density): 0,1

*Optic film added to prevent evaporation

	1	2	3	4	5	6	7	8	9	10	11	12
A	YP 400	YP 400	YP 400	YP 200	YP 200	YP 200	YP 100	YP 100	YP 100	YP 50	YP 50	YP 50
B	YP 25	YP 25	YP 25	YP 12,5	YP 12,5	YP 12,5	YP 0	YP 0	YP 0	YP w/o cells	YP w/o cells	YP w/o cells
C	-	-	-	-	-	-	-	-	-	-	-	-
D	-	-	-	-	-	-	-	-	-	-	-	-
E	YP + sorb 400	YP + sorb 400	YP+ sorb 400	YP + sorb 200	YP + sorb 200	YP + sorb 200	YP 100	YP 100	YP 100	YP 50	YP 50	YP 50
F	YP 25	YP 25	YP 25	YP 12,5	YP 12,5	YP 12,5	YP 0	YP 0	YP 0	YP w/o cells	YP w/o cells	YP w/o cells
G	-	-	-	-	-	-	-	-	-	-	-	-
H	-	-	-	-	-	-	-	-	-	-	-	-

Here is the experiment video.

For more information, check our Results.

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