Difference between revisions of "Yeast Transformation"

From Real Vegan Cheese
Jump to navigation Jump to search
 
(18 intermediate revisions by the same user not shown)
Line 2: Line 2:


In molecular biology, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous DNA (originating outside the cell) from its surroundings through the cell membrane. In yeast, there are numerous ways to perform transformation. One method that is used is Electroporation.  
In molecular biology, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous DNA (originating outside the cell) from its surroundings through the cell membrane. In yeast, there are numerous ways to perform transformation. One method that is used is Electroporation.  
Here is a good summary of he different transformation methods, with their pros and cons:
Here is a good summary of the different transformation methods, with their pros and cons:
[https://www.researchgate.net/profile/R_Gietz/publication/12021077_Genetic_transformation_of_yeast/links/02e7e52ea843b6c2c2000000.pdf Genetic Transformation of Yeast]
[https://www.researchgate.net/profile/R_Gietz/publication/12021077_Genetic_transformation_of_yeast/links/02e7e52ea843b6c2c2000000.pdf Genetic Transformation of Yeast]


Line 16: Line 16:
'''Findings:'''
'''Findings:'''


“Freezing of intact cells in sorbitol with calcium at -80C results in higher transformation efficiency by electroporation, giving more than 106 transformants/μg of plasmid DNA after thawing.”
“Freezing of intact cells in sorbitol with calcium at -80C results in higher transformation efficiency by electroporation, giving more than 10^6 transformants/μg of plasmid DNA after thawing.”
Reference: High-efficiency electroporation by freezing intact yeast cells with addition of calcium, by Suga and Hatakeyama.
Reference: High-efficiency electroporation by freezing intact yeast cells with addition of calcium, by Suga and Hatakeyama.
[http://link.springer.com/article/10.1007%2Fs00294-003-0385-4 High-efficiency electroporation by freezing intact yeast cells with addition of calcium, by Suga and Hatakeyama]




“Pretreatment of yeast cells with lithium acetate (LiAc) and dithiothreitol (DTT) enhances the frequency of transformation by electroporation. The method shows improvements of 6–67-fold in wild-type strains derived from commonly used Saccharomyces cerevisiae genetic backgrounds. In addition, 15–300-fold improvement in transformation frequency was achieved with several mutant strains of S. cerevisiae that transformed poorly by conventional procedures.”
“Pretreatment of yeast cells with lithium acetate (LiAc) and dithiothreitol (DTT) enhances the frequency of transformation by electroporation. The method shows improvements of 6–67-fold in wild-type strains derived from commonly used Saccharomyces cerevisiae genetic backgrounds. In addition, 15–300-fold improvement in transformation frequency was achieved with several mutant strains of S. cerevisiae that transformed poorly by conventional procedures.”
Reference: An improved protocol for the preparation of yeast cells for transformation by electroporation, by Thompson et al.
Reference: An improved protocol for the preparation of yeast cells for transformation by electroporation, by Thompson et al.
[http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0061(19980430)14:6%3C565::AID-YEA251%3E3.0.CO;2-B/epdf]
 
[http://onlinelibrary.wiley.com/doi/10.1002/(SICI)1097-0061(19980430)14:6%3C565::AID-YEA251%3E3.0.CO;2-B/epdf An improved protocol for the preparation of yeast cells for transformation by electroporation]




Line 29: Line 32:
'''Step 1:'''
'''Step 1:'''


Grow S. Cerevisiae cells in YPD medium to a density of approximately 1x107 cells/ml at 30°C.
Grow S. Cerevisiae cells in YPD medium to a density of approximately 1x10^7 cells/ml at 30°C.


Microorganism: S. Cerevisiae cells
Microorganism: S. Cerevisiae cells
Chemicals: SD-URA, Dextrose/Glucose, 5-FOA, Leucine, Agar
Chemicals: YPD
Equipment: Plates (for plate culture) or microfuge tubes (for liquid culture); spectrophotometer od600
Equipment: Plates (for plate culture) or microfuge tubes (for liquid culture); spectrophotometer od600, thermocycler (used for PCR- to control temprature changes at a specific incremental rates)
Temperature req.: 30°C
Temperature req.: 30°C


Line 39: Line 42:


• Place the cultures on ice for 15 min just before harvesting. Collect the cells by centrifugation and wash the resulting pellet three times with ice-cold sterilized water.  
• Place the cultures on ice for 15 min just before harvesting. Collect the cells by centrifugation and wash the resulting pellet three times with ice-cold sterilized water.  
• Suspend this pellet in ice-cold freezing buffer containing 0.6-2.5M sorbitol, 5-10 mM CaCl2, and 10mM of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES, pH=7.5) to give a density of approximately 5x 108 cells/ml
 
• Suspend this pellet in ice-cold freezing buffer containing 0.6-2.5M sorbitol, 5-10 mM CaCl2, and 10mM of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES, pH=7.5) to give a density of approximately 5x 10^8 cells/ml


Microorganism: S. Cerevisiae cells
Microorganism: S. Cerevisiae cells
Line 47: Line 51:
'''Step 3:'''
'''Step 3:'''


Dispense samples (0.1ml) of the cell suspension in 1.5ml microcentrifuge tubes, slowly freeze them and store by placing them directly in a -80C freezer
Dispense samples (0.1ml) of the cell suspension in 1.5ml microcentrifuge tubes, slowly freeze them and store by placing them directly in a -80C freezer. Cooling Rate: 10C/min


Microorganism: cell suspension
Microorganism: cell suspension
Line 58: Line 62:
'''Step 4:'''  
'''Step 4:'''  


• For each electroporation, quickly thaw the frozen competent cells in a water bath at 30C and wash once at with 1ml of ice-cold 1.0 M sorbitol by centrifugation.  
• For each electroporation, quickly thaw the frozen competent cells in a water bath at 30C Warming rate: 200C/min
• Re-suspend the final pellet in 1.0M sorbitol to give a density of 1-2x109 cells/ml.
 
•      Wash once at with 1ml of ice-cold 1.0 M sorbitol by centrifugation.  
 
• Re-suspend the final pellet in 1.0M sorbitol to give a density of 1-2x10^9 cells/ml.


Microorganism: Frozen cells (at -80C)
Microorganism: Frozen cells (at -80C)
Line 68: Line 75:
'''Step 5:'''
'''Step 5:'''


• Mix the cell suspension with 0.5-10mg of purified plasmid DNA and then transfer to a chilled cuvette with a 0.2 cm electrode gap.  
• Mix the cell suspension with 0.5-10ng of purified plasmid DNA and then transfer to a chilled cuvette with a 0.2 cm electrode gap.  
 
• Apply a high electric pulse to the cell suspension, by using the Bio Rad Gene Pulsar II with Pulse Control Plus (or equivalent)
• Apply a high electric pulse to the cell suspension, by using the Bio Rad Gene Pulsar II with Pulse Control Plus (or equivalent)


Line 88: Line 96:
'''Reagents:'''  
'''Reagents:'''  


  SD-URA, Dextrose/Glucose, 5-FOA, Leucine, Agar, Ice, Sterilized water, Buffer, Sorbitol, CaCl2, HEPES with pH 7.5, Plasmid
YPD, Ice, Sterilized water, Buffer, Sorbitol, CaCl2, HEPES with pH 7.5, Plasmid


'''Equipment:'''  
'''Equipment:'''  


  Plates, Spectrophotometer od600, Centrifuge, Pellet, Microcentrifuge tubes, Cuvette (chilled), Electroporation system w/ electrodes, Selection Plates, Dilution equipment (beaker, pipette, etc.…)
Plates, Spectrophotometer od600, Centrifuge, Pellet, Microcentrifuge tubes, Cuvette (chilled), Electroporation system w/ electrodes, Selection Plates, Dilution equipment (tubes, pipette, etc.…), PCR
 
Electric  field strength: 9.5-11KV/cm
 
In the work done by Suga & Hatakeyama, a constant electric pulse of 11kV/cm with 25microF and 200 Ohms was used on a cell concentration of 2x10^9cells/mL

Latest revision as of 17:37, 20 July 2016

What’s Transformation?

In molecular biology, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous DNA (originating outside the cell) from its surroundings through the cell membrane. In yeast, there are numerous ways to perform transformation. One method that is used is Electroporation. Here is a good summary of the different transformation methods, with their pros and cons: Genetic Transformation of Yeast

What's Electroporation?

It’s a technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing DNA to be introduced into the cell.

Important distinctions:

  • Electroporation efficiency = number of transformants per microgram of DNA
  • Electroporation frequency= transformation efficiency per viable cells

Findings:

“Freezing of intact cells in sorbitol with calcium at -80C results in higher transformation efficiency by electroporation, giving more than 10^6 transformants/μg of plasmid DNA after thawing.” Reference: High-efficiency electroporation by freezing intact yeast cells with addition of calcium, by Suga and Hatakeyama.

High-efficiency electroporation by freezing intact yeast cells with addition of calcium, by Suga and Hatakeyama


“Pretreatment of yeast cells with lithium acetate (LiAc) and dithiothreitol (DTT) enhances the frequency of transformation by electroporation. The method shows improvements of 6–67-fold in wild-type strains derived from commonly used Saccharomyces cerevisiae genetic backgrounds. In addition, 15–300-fold improvement in transformation frequency was achieved with several mutant strains of S. cerevisiae that transformed poorly by conventional procedures.” Reference: An improved protocol for the preparation of yeast cells for transformation by electroporation, by Thompson et al.

An improved protocol for the preparation of yeast cells for transformation by electroporation


Preparation (using Suga & Hatakeyama ‘s protocol)

Step 1:

Grow S. Cerevisiae cells in YPD medium to a density of approximately 1x10^7 cells/ml at 30°C.

Microorganism: S. Cerevisiae cells Chemicals: YPD Equipment: Plates (for plate culture) or microfuge tubes (for liquid culture); spectrophotometer od600, thermocycler (used for PCR- to control temprature changes at a specific incremental rates) Temperature req.: 30°C

Step 2:

• Place the cultures on ice for 15 min just before harvesting. Collect the cells by centrifugation and wash the resulting pellet three times with ice-cold sterilized water.

• Suspend this pellet in ice-cold freezing buffer containing 0.6-2.5M sorbitol, 5-10 mM CaCl2, and 10mM of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES, pH=7.5) to give a density of approximately 5x 10^8 cells/ml

Microorganism: S. Cerevisiae cells Chemicals: Ice, Sterilized water, Buffer, Sorbitol, CaCl2, HEPES with pH 7.5 Equipment: Centrifuge, pellet, spectrophotometer od600

Step 3:

Dispense samples (0.1ml) of the cell suspension in 1.5ml microcentrifuge tubes, slowly freeze them and store by placing them directly in a -80C freezer. Cooling Rate: 10C/min

Microorganism: cell suspension Chemicals: Equipment: Microcentrifuge tubes Temperature Req: -80C

Electroporation

Step 4:

• For each electroporation, quickly thaw the frozen competent cells in a water bath at 30C Warming rate: 200C/min

• Wash once at with 1ml of ice-cold 1.0 M sorbitol by centrifugation.

• Re-suspend the final pellet in 1.0M sorbitol to give a density of 1-2x10^9 cells/ml.

Microorganism: Frozen cells (at -80C) Chemicals: Sorbitol, Equipment: Centrifuge, spectrophotometer od600


Step 5:

• Mix the cell suspension with 0.5-10ng of purified plasmid DNA and then transfer to a chilled cuvette with a 0.2 cm electrode gap.

• Apply a high electric pulse to the cell suspension, by using the Bio Rad Gene Pulsar II with Pulse Control Plus (or equivalent)

Microorganism: Cell suspension Chemicals: Equipment: Cuvette (chilled), Electroporation system w/ electrodes Other: plasmid

Step 6:

Immediately dilute the electroporated cells in 1ml of ice-cold 1.0 M sorbitol and spread samples (0.1-0.2 ml) on minimal selection plates. Note: The sorbitol acts as an osmotic stabilizer

Microorganism: Electroporated Cells Chemicals: Sorbitol Equipment: Dilution equipment, , Selection Plates

Summary of Reagents & Equipments Need to Complete the above protocol

Reagents:

YPD, Ice, Sterilized water, Buffer, Sorbitol, CaCl2, HEPES with pH 7.5, Plasmid

Equipment:

Plates, Spectrophotometer od600, Centrifuge, Pellet, Microcentrifuge tubes, Cuvette (chilled), Electroporation system w/ electrodes, Selection Plates, Dilution equipment (tubes, pipette, etc.…), PCR

Electric field strength: 9.5-11KV/cm

In the work done by Suga & Hatakeyama, a constant electric pulse of 11kV/cm with 25microF and 200 Ohms was used on a cell concentration of 2x10^9cells/mL