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The following protocol is a slight modification of the “Quick and Dirty” transformation protocol described by Amberg et al. (2005). Modifications to this method can increase its efficiency by several orders of magnitude (Gietz and Schiestl, 2007), which would be required if linear pieces of DNA were being used to tranform yeast.
Prepare a transformation master mix
1. Prepare a transformation master mix. The following ingredients provide enough reagents for five transformation reactions. Combine and mix in a microcentrifuge tube:
100 μL sterile 2 M lithium acetate (freshly prepared)
400 μL sterile 50% PEG-3350
4 μL 2-mercaptoethanol (STINKY!! add this in the fume hood!)
Set up individual transformation reactions - for each transformation:
2. Add 15 μL of the denatured salmon sperm DNA (2 mg/mL) to a new microcentrifuge tubelabeledwith the name (or code) of the plasmid.
Note: It is important for the salmon sperm DNA to be single-stranded for this procedure to work well. Boil the DNA for 5 minutes to denature the DNA. Quick chill the DNA by placing it immediately on ice. Keep the DNA on ice until you are ready to use it.
3. Add 5 μL of miniprep plasmid DNA to the appropriately labeled microcentrifuge tube.
4. Add 100 μL of transformation mix from step 1 to each microcentrifuge tube. Vortex for 10-15 seconds to mix the contents.
5. Using a sterile toothpick or micropipette tip, scrape a large yeast colony (or the equivalent of a “match head” of yeast) from a YPD plate. Transfer the yeast to the microcentrifuge tube containing the transformation/DNA solution (step 4) by twirling the toothpick several times. Be sure that the cells are uniformly suspended before proceeding.
Repeat steps 2-5 for each of your transformation reactions. Be sure to include a control that contains no plasmid DNA.
6. Incubate the transformation mixtures at 37 ̊C with shaking for 30-45 minutes.
Plate the transformed cells on selective media lacking uracil.
7. Remove 10 μL of the resuspended cells and add them to 90 μL of sterile water in a microcentrifuge tube. This sample will be serially diluted for a spot plate (step 9) that you will use to calculate the transformation efficiency.
8. Spread the remainder of the mixture on a selective media plate lacking uracil. Transfer the transformation reaction to the plate, and then shake out ~4 sterile glass beads that will spread the cells. Cover the plates and spend 0.5-1 minutes agitating the plates so that the beads spread the transformation mixture evenly over the surface of the plate. Discard the glass beads into the appropriate waste containers, so they can be sterilized and used again. Incubate the plates at 30 ̊C until colonies can be detected. The earliest that colonies will be visible is usually 2 days. If the colonies are small, allow them to grow an additional day(s) at 30 ̊C. Count the number of colonies on the plate.
Determine the number of viable cells in the transformation mixture.
9. Prepare a series of 4 additional dilutions of the cells set aside in step 7. Use these dilutions
for a spot plate on YPD media. Each row on the plate should contain cells from a different transformation reaction. Incubate the cells at 30 ̊C or room temperature until individual colonies can be detected. Do not allow the plate to overgrow, because you need to distinguish individual colonies.
Calculate the transformation efficiency. The efficiency of transformation is influenced by both the quality of the DNA used and the precise details of the transformation procedure.
10. Calculate the fraction of cells that were transformed as shown below. The total volume of transformation mixture was ~120 μL, including yeast cells. Ten μL was used for spot plating and the remaining 100 μL was used for the transformation.
11. Transformation efficiencies are usually expressed by the number of cells transformed per μg DNA. In the last lab (Chapter 11), you analyzed your plasmid preparations on agarose gels and obtained a rough estimate of the DNA concentrations of your plasmid preparations. Note that you analyzed 7 μL of plasmid prep on those gels. In this transformation lab, you used 5 μL of your plasmid preps.
Calculate the transformation efficiency:
A. Multiply that number of transformed cells on the YC-plate by 1.1
(Only 100 out of 110 μL in the transformation reaction were plated.)
B. Convert the ng of plasmid in your transformation reaction to μg.
C. Divide the calculated value in A by that in B.
Development of synthetic biology tools to engineer Pichia pastoris as a chassis for the production of natural products
The methylotrophic yeast Pichia pastoris (a.k.a. Komagataella phaffii) is one of the most commonly used hosts for industrial production of recombinant proteins. As a non-conventional yeast, P. pastoris has unique biological characteristics and its expression system has been well developed. With the advances in synthetic biology, more efforts have been devoted to developing P. pastoris into a chassis for the production of various high-value compounds, such as natural products. This review begins with the introduction of synthetic biology tools for the engineering of P. pastoris, including vectors, promoters, and terminators for heterologous gene expression as well as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated System (CRISPR/Cas) for genome editing. This review is then followed by examples of the production of value-added natural products in metabolically engineered P. pastoris strains. Finally, challenges and outlooks in developing P. pastoris as a synthetic biology chassis are prospected.
Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology
Transformation-associated recombination (TAR) cloning represents a unique tool for isolation and manipulation of large DNA molecules. The technique exploits a high level of homologous recombination in the yeast Sacharomyces cerevisiae. So far, TAR cloning is the only method available to selectively recover chromosomal segments up to 300 kb in length from complex and simple genomes. In addition, TAR cloning allows the assembly and cloning of entire microbe genomes up to several Mb as well as engineering of large metabolic pathways. In this review, we summarize applications of TAR cloning for functional/structural genomics and synthetic biology.
Keywords: HAC Human artificial chromosome Synthetic biology TAR cloning Transformation-associated recombination.
Conflict of interest statement
The authors declare that they have no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors.
This protocol was submitted by Matt Kaeberlein.
This protocol works well for transforming plasmids and PCR disruption cassettes.
- TE + 100mM LiAc (Dilute stock 1M LiAc and 10X TE in water 1:1:8).
- PLATE (1X TE, 100 mM LiAc 40% PEG)
- Grow yeast to be transformed overnight in YPD.
- Dilute back overnight culture 1:100 in fresh YPD and culture at 30C for 4-6 hours.
- Spin down 15mL of cells.
- Wash cells 2X in 0.5mL TE + 100 mM LiAc
- Resuspend cells in 200uL TE + LiAc
- Add transforming DNA.
- Add 20uL freshly boiled ssDNA.
- Add 1mL PLATE solution. Mix by inversion several times.
- Incubate at 30C for 30 min.
- Incubate at 37C for 15 min. Mix by inversion every 5 min.
- Spin down cells and plate on selective media.
For transforming DNA I use 1 uL of mini-prep DNA for a plasmid and 30-50uL of PCR product for a PCR disruption.
In practice, I usually scale up and spin down 45 mL of cells in a 50 mL tube. I do the 2X washes then split the cells into 3 individual eppendorf tubes for transformation.
Agrobacterium-mediated plant transformation: biology and applications
Plant genetic transformation heavily relies on the bacterial pathogen Agrobacterium tumefaciens as a powerful tool to deliver genes of interest into a host plant. Inside the plant nucleus, the transferred DNA is capable of integrating into the plant genome for inheritance to the next generation (i.e. stable transformation). Alternatively, the foreign DNA can transiently remain in the nucleus without integrating into the genome but still be transcribed to produce desirable gene products (i.e. transient transformation). From the discovery of A. tumefaciens to its wide application in plant biotechnology, numerous aspects of the interaction between A. tumefaciens and plants have been elucidated. This article aims to provide a comprehensive review of the biology and the applications of Agrobacterium-mediated plant transformation, which may be useful for both microbiologists and plant biologists who desire a better understanding of plant transformation, protein expression in plants, and plant-microbe interaction.
Major steps of the Agrobacterium…
Major steps of the Agrobacterium tumefaciens-mediated plant transformation process. (1) Attachment of A.…
Yeast transformation - low efficiency (Dec/15/2008 )
I transform the yeast using LiOAc/PEG method. The efficiency was low, about 10^3-10^4 colonies /ug plasmid.
The protocol was:
240 ul 50% w/v PEG3350
35 ul 1M LiOAc
25 ul carrier ssDNA
1 ul plasmid
Mix well and add to the washed cells.
30 degree C, 30 minutes
42 degree C, 20 minutes
spin down, resuspend in water
what kind of yeast are you working with?
If this is S.pombe, I would say you should increase the duration of the 30 C incubation to 1hr, reduce the heat shock (42 C) to around 10-5min (I personally use 5min) and add 42ul DMSO to improve the heatshock.
Also how hard are you spinning your cells? Spin for the shortest time possible at the lowest speed setting your machine can go. The yeast cells are fragile after the transformation protocol.
If you do add the DMSO, wash it off before plating the cells.
Once your cells are plated, keep your incubator humidified. S.pombe does not like dried plate surfaces. This helps improve growth rate and to a lesser extent recovery rate. So if you do use humidity and are colony counting make sure you do this for all your experiments.
Could you tell me abit more about what you do once your cell culture is ready. Do you wash in your cells in TE+LiAc before making a final DNA+cell mix?
Lastly the LiAc transformation protocol is a bit crude and can perhaps be pushed to around 10^5 colonies/ug DNA but not much more. There are better protocols, but are more complex and time consuming.
Thank you, perneseblue,
I am working with baker's yeast. The cells were pelleted by cfg at 3000 rpm, 5 minutes. Washed once with 1x volume water. The transformation mix( plamsid, ssDNA, PEG3350, LiOAc) was then added to the cell pellet. Mixed by vortexing for 10 seconds. After 30 minutes 30 degree C, and 20 minutes 42 degree C incubation, the cells were pelleted, resuspended in water, and plated on selection plates.
I will try to add DMSO next time.
This protocol is based on Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96. Some steps of this protocol are based on information obtained from:
This protocol is generalized and not to be considered optimal for all strains, conditions, and applications. Some aspects of this protocol may have to be modified significantly to suit your particular needs. It works for simple transformation, targeting in yeast, GAP repair, and multi-plasmid transformations for two-hybrid.
1) Streak or patch yeast on YAPD for 24-48hrs. Can also use yeast from older plates (3 months? at 4 deg) if efficiency is not an issue and the strain cooperates.
2) Inoculate 2 x 3-5ml cultures in 2xYAPD or SC media (15ml snap cap tubes) from the fresh streak and incubate O/N 30 deg, 200rpm. SC media may need to be seeded more heavily (or more tubes may be required).
YAPD can be substituted for 2xYAPD but efficiency will drop some and yeast will take longer to grow.
1) Pre-warm 50ml 2xYAPD to 30deg in 500ml flask (at least 10min). Turn on 42deg water bath (takes 30min or more).
2) Dilute O/N culture 1:10 in water and take an OD at 600nm. Use 2xYAPD (or YAPD or SC when appropriate) diluted 1:10 in water as a blank. The diluted culture will usually have an OD of between 0.1 and 1.0.
3) Dilute culture to approximately 0.1 OD in 50 ml pre-warmed 2xYAPD and incubate 3-6hrs until OD reaches 1.0 (or close). Yeast grown rates vary so be prepared to wait. It usually takes 5-6 hours but can take quite a bit longer.
EXAMPLE: The diluted culture in step 2 has an OD or 0.2 so the culture OD is 2.0. To get an OD of approximately 0.1 for step 3 you would add 2.5ml of culture to your pre-warmed 50ml 2xYAPD (20x dilution). Obviously its not exact because you will now have 52.5ml instead of 50ml but its close enough. If you want it exact you can simply remove 2.5ml 2xYAPD from the flask before you add your 2.5ml culture but its not necessary.
4) Pellet 3000rpm, 10min at RT in RT6000 or similar centrifuge.
5) While spinning yeast, boil salmon sperm DNA 5 min with boiling cap and chill on ice. Use 3-4 times (storing at -20) and get a new tube. Do not boil over and over again.
6) Resuspend in 1ml ddH2O and transfer to 1.5ml tube.
7) Vortex 1min or until yeast are in suspension (no clumps).
8) Pellet at max speed, 30sec, RT and resuspend in 1ml ddH2O by vortexing as before. 100ul will be used for each transformation so there are enough yeast for 10 transformations.
9) Transfer 100 ul of yeast to a separate 1.5ml tube for each transformation and pellet at max speed, 30sec, RT and remove supernatant.
10) For each transformation make up the following (master mix or separately):
50ul of 2mg/ml salmon sperm DNA
34ul or your plasmid(s) + water (1ul of bacterial miniprep DNA is plenty)
11) Add the transformation mix(es) to the yeast pellets from step #9 and vortex 1min or until yeast are in suspension (no clumps).
12) Place yeast at 42 deg for 40min. For best efficiency the optimal shock time needs to be determined empirically and can vary from 10min to 60min. For simple single selection plasmid transformation 20-30min usually works fine.
13) Pellet at max speed, 30 sec, RT and remove transformation mix.
14) Add 1ml ddH2O and use a 1ml pipet tip to stir the cells into solution. If necessary, pipet the yeast up and down very gently.
15) Plate 200ul on appropriate 100mm or 150mm selective plates and grow 3-4 days at 30deg. *Instead of 1ml in step 14 you can also resuspend the yeast in 400ul and plate the entire amount on one 150mm plate if you desire.
Efficiency varies greatly based on strain and transforming DNA. It can vary from 1吆^3 to 1吆^5 (or so) colonies per ug of input DNA (for plasmid DNA).
10g of BactoYeast extract
100mg adenine hemisulfate
Autoclave 20min on liquid cycle.
Selective medium will vary based on strain and application.
2mg/ml in TE. Sigma D1626. Make 100ml in a flask using a magnetic stirrer. Takes 2-4 hours to dissolve. Aliquot in 1.5ml tubes (1ml/tube).
Place 50gm of polyethylene glycol, MW 3350 (Sigma) in a 150 ml glass beaker and add 35 ml of ddH20. Stir for 1-2hrs with a magnetic stirring bar until dissolved. q.s. to 100 ml and keep stirring for at least 10min to mix. Filter using a 0.45 um filter and aliquot in 15ml conicals (10ml each) and store at RT.
1.0M Lithium Acetate. No need to pH.
Tim Schedl, Ph.D
Department of Genetics
Campus Box #8232
Washington University School of Medicine
4566 Scott Ave.
St. Louis, MO 63110