Team:USP-Brazil/LaBioTec

From 2013.igem.org

(Difference between revisions)
Line 11: Line 11:
<h2>LaBioTec</h2>
<h2>LaBioTec</h2>
-
<p>Texto aqui</p>
+
<p><b>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 Paox1 amplification and determinate optimum temperature and DNA template concentration
 +
        T<sub>melting</sub>: 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.
 +
To Growth curve measure it was used a cuvette measuring 9 points at all, with two initial ODs:
 +
*COLOCAR OS GRAFICOS QUE TAO NO BIOTEC LAB NOTEBOOK*
 +
Figure 5: growth curve of Pichia pastoris. Gray x mark the actual data; colored circles represents the mean and the line is the fitted logistic curve as shown in Equation 1.
 +
 
 +
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
 +
 
 +
We achieved the following results for the two initial Pichia pastoris cultures:
 +
Table 1
 +
(COLOCAR OS GRAFICOS QUE TAO NO DRIVE)
 +
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 eppendorfs and in small flasks, well mixed, fast frozen with liquid nitrogen, opened and put under lyophilization for 24 hours
 +
Table 2
 +
After lyophilization, the 4 different samples were suspended in 1 mL water and serially diluted  from 10¹ to 10⁸ 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.
 +
Pre Lyophilization inoculum results (the number of CFU of each resuspension drops is in parenthesis):
 +
Table 3
 +
 
 +
10 July - 19 July
 +
1 mL of inoculum was centrifuged and resuspended in 500 uL(“X”). The same were made with 2 mL of inoculum.(“2X”)
 +
3 replicates of the first resuspended inoculum (“X”) was mixed with 250 uL of milk solution and 250 uL of monosodium glutamate solution(1,2 e 3). The same was made with the second inoculum (2X), but in only one sample.
 +
(NAO TA MUITO CLARO ACIMA!)
 +
All samples were lyophilized for 24 hours. A sample of 1 mL of the inoculum was plated under serial dilutions from 10¹ to 10⁸.
 +
The lyophilized samples were resuspended  in 1 mL of water, serially diluted from 10¹ to 10⁶ 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 wasn’t lyophilized (control) was plated under serial dilutions from 10³ to 10⁸
 +
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,
 +
And one in 20% Ethanol solution,
 +
and serially diluted from 10¹ to 10⁸ and plated from 10³ to 10⁸. 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 lyophilisation preparation
 +
Started the lyophilization process again with a new milk solution.
 +
 
 +
 
</div>
</div>

Revision as of 23:12, 27 September 2013

Template:Https://2013.igem.org/Team:USP-Brazil/templateUP

Notebook

LaBioTec

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 Paox1 amplification and determinate optimum temperature and DNA template concentration Tmelting: 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. To Growth curve measure it was used a cuvette measuring 9 points at all, with two initial ODs: *COLOCAR OS GRAFICOS QUE TAO NO BIOTEC LAB NOTEBOOK* Figure 5: growth curve of Pichia pastoris. Gray x mark the actual data; colored circles represents the mean and the line is the fitted logistic curve as shown in Equation 1. 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 We achieved the following results for the two initial Pichia pastoris cultures: Table 1 (COLOCAR OS GRAFICOS QUE TAO NO DRIVE) 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 eppendorfs and in small flasks, well mixed, fast frozen with liquid nitrogen, opened and put under lyophilization for 24 hours Table 2 After lyophilization, the 4 different samples were suspended in 1 mL water and serially diluted  from 10¹ to 10⁸ 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. Pre Lyophilization inoculum results (the number of CFU of each resuspension drops is in parenthesis): Table 3 10 July - 19 July 1 mL of inoculum was centrifuged and resuspended in 500 uL(“X”). The same were made with 2 mL of inoculum.(“2X”) 3 replicates of the first resuspended inoculum (“X”) was mixed with 250 uL of milk solution and 250 uL of monosodium glutamate solution(1,2 e 3). The same was made with the second inoculum (2X), but in only one sample. (NAO TA MUITO CLARO ACIMA!) All samples were lyophilized for 24 hours. A sample of 1 mL of the inoculum was plated under serial dilutions from 10¹ to 10⁸. The lyophilized samples were resuspended  in 1 mL of water, serially diluted from 10¹ to 10⁶ 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 wasn’t lyophilized (control) was plated under serial dilutions from 10³ to 10⁸ 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, And one in 20% Ethanol solution, and serially diluted from 10¹ to 10⁸ and plated from 10³ to 10⁸. 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 lyophilisation preparation Started the lyophilization process again with a new milk solution.

Template:Https://2013.igem.org/Team:USP-Brazil/templateDOWN