Protocol for Yeast Recombinational Cloning
A map of a capture vector used to clone genomic DNA in yeast is depicted in figure 1.
Construction of the capture vector:
The capture vector is assembled using homologous recombination in yeast. There are four fragments that are needed: upstream and downstream targeting sequences homologous to the borders of the desired fragment to be cloned (1kb each) generated by PCR, the vector, pLLX13, that contains components necessary for 1) the capture vector's selection in yeast, E. coli, and P.aeruginosa, 2) replication in yeast and bacteria, and 3) its transfer, and a PCR product amplified from pLLX8 containing an ampicillin resistance gene and a counterselection against the growth of yeast (CYH2) in the presence of cycloheximide.
Alternatively, pLLX16Kan can be used instead of pLLX13. This vector is not able to replicate in P. aeruginosa.
A diagram of the four fragments and how they will recombine to form the capture vector is included in figure 2.
Primer design for the targeting sequences:
The upstream and downstream targeting sequences that have been used are one kb each in length and are designed to be the sequences bordering the DNA region that is intended to be cloned. They are generated by PCR and homologies to facilitate recombination necessary for assembly of the capture vector are added to the upstream and downstream targeting sequences by adding tails to the 5' end of each primer. For the upstream targeting sequence, the extension to the sense primer, named P1, is: 5'-ATA TTA CCC TGT TAT CCC TAG CGT AAC TAT CGA TCT CGA G-3'. This sequence provides 40 bp of homology to pLLX13.
The tail added to the antisense primer, named P2 is:5'-CAT ATA TAC TTT AGA TTT TAA TTA AAC GCG TTC TAG AAA A-3' This sequence provides 40 bp of homology to the pLLX8 PCR.
For the downstream targeting sequence, the tail added to the sense primer, named P3, is: 5'-CAT TTT CAC CGT TTT TTG TTT AAA CGT TAA CTC TAG AGG G-3' This sequence provides 40 bp of homology to the pLLX8 PCR.
The tail added to the antisense primer, named P4, is: 5'-TAA CAG GGT AAT ATA GAG ATC TGG TAC CCT GCA GGA GCT C-3' this sequence provides 40 bp of homology to pLLX13.
Preparation of the fragments:
PCR the plasmid pLLX8 with the primers: 5'- TTT TCT AGA ACG CGT TTA ATT AAA ATC TAA AGT ATA TAT GAG TAA AC-3'
and
5'-C CCT CTA GAG TTA ACG TTT AAA CAA AAA ACG GTG AAA ATG GGT GAT AG-3'
the resulting 2.95 kb fragment will contain the bla and CYH2 genes
To generate targeting sequences, use the two sets of primers designed according to the instructions above.
Gel purify the PCR products.
Linearize the plasmid pLLX13 DNA with the enzyme NheI:
resulting in a 9.940 kb fragment.
Heat kill the enzyme from the digest.
You will need approximately 200 ng each of the upstream and downstream homology fragments, 600 ng of the pLLX8 PCR and 200 ng of the pLLX13 digest per transformation of yeast. These amounts do not need to be exact. The assembly is usually very efficient.
The next step is to cotransform all four of the fragments into competent S. cerevisiae strain CRY1-2 which is resistant to cycloheximide.
Transformation protocol:
Streak onto a YPD plate strain CRY1-2. This can be reused for 1-2 months.
Inoculate a few colonies into 50ml of YPD. Grow in a 250ml baffled flask shaking at 250 rpm at 30 degrees Celsius overnight. After 12-16 hrs inoculate the desired amount of YPD (every 50 ml gives approximately 1.5 transformations) with a dilution of approximately 1:100 and adjust the final OD 600 so it is approximately 0.05. Let the culture grow for around five hours with shaking at 30 degrees until the final OD is 0.4-0.5. Transfer the culture to 50ml conical tubes and centrifuge at room temperature for 5 minutes at 1,200 rcf.
Discard the supernatant and resuspend in 40 ml sterile RT water. Spin again at room temperature for 5 minutes at 1,200 rcf.
Discard the supernatant and resuspend in 250 microliters TFBI. Add the DNA to a 5 ml snap cap tube along with 200 micrograms of single stranded herring sperm DNA. pLLX13 should be transformed as a positive control. Then add 200 microliters of the yeast suspended in TFBI to the 5 ml tube followed by 1.2 ml of TFBII and vortex until homogeneous.
Put the tubes in a 30 degree water bath for 30 minutes followed by a 42 degree water bath for 15 minutes. Then pellet the cells for 2 minutes at 1,200 rcf, dump the supernatant, and gently resuspend (no vortexing) in 500 microliters TE. Plate 100 microliters onto uracil dropout plates and put in a 30 degree incubator. Colonies will appear in 2-3 days. There should be hundreds of colonies per transformation.
To obtain the desired plasmid, purify DNA from the transformants as a pool. Swab each plate, resuspend the yeast in PBS, pellet the cells and then use a Zymoprep yeast plasmid miniprep kit to purify the yeast DNA. Then electroporate into E.coli DH10B (high-efficiency is required) selecting for 5 micrograms/ml tetracyline and 100 micrograms/ml ampicillin resistance if pLLX13 was used or 50 micrograms/ml kanamycin and ampicillin if pLLX16kan was used. Pick colonies and analyze for the desired plasmid. After selection on ampicillin and tetracycline or kanamycin, usually greater than 90% of the plasmids analyzed are correct.
When the correct plasmid has been identified, perform a test transformation of yeast to verify the cycloheximide counterselection works. Select on both uracil dropout media with and without cycloheximide 2.5 micrograms/ml. The rate of reversion for cycloheximide sensitivity should occur in less than 1% of the transformants. It is around 10 percent for linearized plasmids.
Recombinational Cloning:
Linearize the vector DNA with PmeI or MluI.
If the size of the piece of DNA to be cloned is less than or equal to 30 kb, then the previous method of preparing yeast competent cells is recommended. Per transformation, use 500 ng of linearized vector and 5 micrograms of sheared genomic DNA (passed through a 26 1/2 gauge needle 40 times).
Transform the capture vector as a positive control to test the transformation efficiency.
Select for capture vectors containing genomic DNA by plating the transformation on uracil dropout plates with the addition of cycloheximide. Expect 0 to 100 colonies per transformation.
In my experience, 2% to 10% of colonies have plasmids with the genomic DNA that was desired to be cloned.
If the size of the DNA to be cloned is greater than 30 kb, then the spheroplast method of preparing competent yeast may be the only way to clone the DNA of interest. I am not sure about this but from the lab's experience, 30kb fragments are easily cloned using chemically competent yeast but fragments 85 kb in length have never been successfully cloned using chemically competent yeast and have only been cloned by using spheroplasts.
Transformation protocol for preparing spheroplasts:
Innoculate 50 ml of YPD as described before, dilute 1:100 and grow to OD 600 of 0.5 to 0.8. Transfer the culture to 50 mL conical tubes and spin at 1,200 rcf for 5 minutes at room temperature.
Wash one time in 40 mLs sterile water and centrifuge as above.
Repeat using 20 mLs 1 M sorbitol.
Resuspend in 20 mLs SCE.
Remove 100 microliters for future use.
Add 200 microliters 1 M DTT and 50 microliters Zymolyase 20T (10mg/ mL in 10mM sodium phosphate buffer pH 7.5)
Place tubes in a 30 degree water bath.
Monitor for spheroplast formation by adding 100 microliters of yeast to 900 microliters of water and compare the OD 800 reading to the non spheroplast sample that was removed earlier. When the OD 800 reading is 10% of the original, remove from the water bath.
At this point, it is necessary to be much more gently with the cells because they are more fragile due to the digestion of the cell wall.
Centrifuge at 300 rcf for 4 minutes at room temperature.
Resuspend in 20 ml 1 M sorbitol.
Wash as above using 20 ml STC.
Resuspend in 2 ml STC.
Per transformation gently mix 200 microliters yeast, 1 microgram linearized plasmid, 10 micrograms of unsheared genomic DNA, and 5 micrograms of single stranded herring sperm DNA all to a 5 ml snap cap tube.
Always include a positive control for the transformation such as the capture vector or pLLX13.
Let sit at room temperature for 10 minutes.
Add 2 ml PEG, gently mix, and incubate for 10 more minutes.
Centrifuge at 300 g for 5 minutes and gently resuspend in 150 microliters SOS.
Incubate at 30 degrees for 30 minutes.
Add to 8 ml top agar at 46 degrees and pour onto a 1M sorbitol uracil dropout cycloheximide 2.5 micrograms/ml plate.
Colonies from the positive control take two days to appear. Colonies from the recombinational cloning can take three days to appear.
To Screen colonies for correct insert:
Patch first onto the same selective media as previously used. This is necessary because you can get false positives from the PCR screening below if the colony isn't patched before using for PCR due to residual genomic DNA on the plate.
Create spheroplasts so the cells may be lysed:
Resuspend a small amount of yeast from each patch into 10-15 microliters spheroplast solution.
Incubate at 37 degrees with shaking for 1hr.
PCR to detect plasmids containing genomic DNA:
Add 1 microliters of the yeast spheroplasts into a 25 microliter reaction.
For the PCR program, incubate for five minutes at 94 degrees celsius and use 40 cycles of denaturing, annealing, and extension.
Design your own controls for yeast lysis and PCR are very important as this step is prone to error.
Upon identifying the patch containing the plasmid with the correct insert, resuspend the patch in PBS and pellet. Isolate DNA as previously described and electrotransform one microliter into E. coli DH10B. Select on L Agar with 5 micrograms/ml tetracycline (pLLX13) or 50 micrograms/ml kanamycin (pLLX16kan).
Upon obtaining colonies, perform another PCR to verify that the desired plasmid is still present.
To purify plasmid DNA, perform a Qiagen MIDI or MAXI prep.
Solutions and Media:
TFBI
1 volume 1M LiAcetate
1 volume 10X TE pH7.5
8 volumes water
TFBII
1 volume 1M LiAcetate
1 volume 10X TE
8 volumes 50% PEG 3350 (Sigma)
Uracil Drop out Plates/ Liter
20 g agar
800 ml water
Autoclave and
Add 200 ml sterile 5X (per liter) Minimal SD media (BD Biosciences Clontech Cat# 630411/8602-1)+ Uracil Dropout supplement (Clontech Cat# 8607-1)
Top Agar
2.5% Agar
1M Sorbitol
STC
1 M Sorbitol
10 mM tris, pH 7.5
10 mM CaCl2
SCE
1 M Sorbitol
100 mM NaCitrate pH 5.8
10 mM EDTA pH 8
PEG
20% PEG 8000 (Sigma)
10 mM Tris base (pH 7.5)
10 mM CaCl2
SOS
25 ml YPD
700 microliters 1M CaCl2
18.2g sorbitol
water qs to 100 ml
Spheroplast solution:
Make a stock concentration of Zymolyase 20T 25 mg/ml dissolved in 10mM sodium phosphate buffer pH 7.5 and 1M sorbitol. Freeze at -80 C in 100 microliter aliquots
Dilute 1:10 in the same solution it was dissolved in for use that contains 10mM DTT.
Supplies:
Zymolyase-20T:
ICN cat#320921
Cycloheximide:
Obtained from Sigma, cat# C7698
Stock solution is 10 mg/ml
Dilute 1:4000 to make 2.5 micrograms/ml
Single stranded herring sperm DNA:
Clonetech cat# K1606A (yeast maker carrier DNA)
Upon receipt, boil for 5 minutes and then place on ice to ensure it is single stranded, and then aliquot into appropriate amounts
Zymoprep, Yeast Plasmid Miniprep kit is obtained from Zymo Research cat# D2001
Electrocompetent E.coli:
Invitrogen electrocompetent E. coli DH10B 18290-015
Invitrogen has a variant of DH10B called GeneHogs (cat# C8080) that have been modified to take up large plasmids.
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