A rapid method for generating gene deletions in Aspergillus

Thursday, November 13, 2003

Description
A rapid method for generating gene deletions in Aspergillus fumigatus

Procedure
Step 1: Introduction of target cosmid into KS272(pKOBEG)
The E. coli strain KS272(pKOBEG) is transformed with the relevant target cosmid according to standard E. coli procedures (i.e., either electro-competent or CaCl2-competent E. coli cells can be used). It is advisable to use a selectable marker other than CmR or ZeoR. Transformants must be selected at 30 C and LB agar plates should include both chloramphenicol (25 g/ml) and the relevant antibiotic for the target cosmid (e.g., ampicillin or kanamycin (50 g/ml)). A transformant is selected which carries both the plasmid pKOBEG and the target cosmid. This transformant is used for making electrocompetent cells.

In our case we used a chromosomal A. fumigatus cosmid library (Langfelder et al., 1998) derived from the plasmid pANsCos1 (Osiewacz, 1994).

Step 2: Amplification of the bi-functional marker
PCR products are generated using the plasmid pCDA21 as template (other templates are possible). The primers used have two distinct regions.

The 5 region (50-60 bp) corresponding to the sequence on the cosmid where the recombination event should take place (e.g., upstream of the relevant gene).

The 3 region (approximately 20 bp) which is homologous to the peripheral region of the bi-functional marker (i.e., either to zeoR or pyrG).

ZEO-primer: 5-N55X:GGAATTCTCAGTCCTGCTCC-3

PYRG-primer: 5-N55Y:GAATTCGCCTCAAACAATGC-3

These two primers are oriented in opposite directions and bind at the outside ends of the zeoR and pyrG genes, respectively, so that a 2.7 kb PCR product results. This PCR product also includes the 50-60 bp of homologous sequence required for the recombination event. The flanking sequences (denoted by N55) should be chosen to lie outside the region to be deleted and should also be oriented in opposite directions (i.e., pointing towards each other).

Proposed PCR program:

93 C 5
93 C 30
58 C 2
72 C 3 repeat steps 2-4, 4x
93 C 30
60 C 2
72 C 3 repeat steps 5-7, 24x
72 C 10
In 100 l: 200 ng of pCDA21, 25 nmol dNTPs, 100 pmol primers, 5 U Taq DNA polymerase.

Step 3: Recombination
The PCR product is dialysed twice on a Millipore 0.025 m filter floated on distilled water. 10 l of this solution are used to transform electrocompetent E. coli cells (KS272(pKOBEG + target cosmid)).

Method for making electrocompetent cells.

Materials:

500 ml LB medium in 1 l flask
2 l of ice-cold, sterile dH2O
50 ml of ice-cold, sterile 10 % [v/v] glycerol solution
sterile Eppendorf cups

Inoculate a 5 ml overnight culture of the strain containing both plasmid pKOBEG and the target cosmid at 30 C, Cm (25g/ml) + Amp (50g/ml), 180 rpm. Use 2.5 ml of this culture to inoculate 500 ml of LB medium containing Cm (25g/ml) + Amp (50g/ml) + 0.2 % [w/v] arabinose. Shake at 30 C and 180 rpm until the culture reaches OD600 0.5-0.8.

Centrifuge cells in a cold rotor at 4000 x g for 10 minutes. Remove supernatant.
Resuspend cells in 500 ml cold dH2O and centrifuge again (4000 x g, 10 minutes). Repeat 3 times. Keep cells on ice at all times.
Remove supernatant and resuspend cells in 40 ml cold dH2O. Centrifuge as above (in 50 ml corning tube).
Remove supernatant and gently resuspend cells in 1.5 ml 10 % [v/v] ice-cold glycerol solution (final cell concentration should be around 1010 cells/ml).
Freeze 50 l aliquots in liquid nitrogen. Store at 70 C for up to 6 months.
One aliquot can be used for electroporation using 10 l of the dialysed PCR product. The conditions for electroporation are as follows: 200 Ohm, 25 F and 2.5 kV (the time constant should be around 4.5 ms). After electroporation cells should be incubated in 1 ml LB medium for 30 minutes (30 C) before plating on relevant agar plates (LBLS zeocin (50 g/ml) + relevant antibiotic, ampicillin (50 g/ml) in our case). Plates are incubated at 30 C. Transformants appear within 24 hours.

Transformants can be grown at 37 C (LBLS zeocin + e.g., ampicillin) in order to cure them of the plasmid pKOBEG (which is temperature sensitive). They should contain the target cosmid in which the relevant gene/region has been replaced by the bi-functional marker. This can be verified by restriction digest (sometimes inconclusive because of the large number of fragments), PCR or sequencing.

Two primers which are directed outwards from the bi-functional marker can be used in conjunction with primers which lie on the cosmid to generate PCR products (these can also be sequenced in order to check for correct integration).

ZEOinternal 5-GTGACCCTGTTCATCAGC-3

PYRinternal 5-TCCACGGAACTTTTAACG-3

Once a correct cosmid (i.e., one in which the relevant gene has been replaced by the bi-functional marker) has been identified, this can be used for transformation of A. fumigatus. The circular cosmid can be used or it can be digested with an appropriate restriction enzyme, followed by dephosphorylation. This will increase the probability of a gene deletion occurring and decrease the number of single recombination events see figure 2.

In our case the circular cosmid was used to transform the A. fumigatus CEA17 strain. 20 transformants resulted from the first transformation. Of these 8 were analysed by Southern blot analysis and 3 were found to have a deletion of the relevant gene. It is possible to preselect transformants resulting from homologous recombination since these should still be sensitive to hygromycin B. In the case of an ectopic integration of the cosmid (which carries the hph gene and hence confers hygromycin B resistance) the transformants should be resistant to hygromycin B.

If a cosmid containing the relevant gene/region is available then this method can be used to generate deletion strains very rapidly and without much DNA manipulation. The large flanking regions result in a high percentage of A. fumigatus transformants with the relevant gene deletion. The critical step in this procedure is the quality of electrocompetent cells. Varying the arabinose concentration can increase the recombination efficiency as can an increase in the amount of PCR product used in the transformation.

Some cosmids may not be suited to this recombination system although it is not yet clear why this should be the case. Cosmids derived from pWE15 appear to work while LORIST2 cosmids have not done so to date.

Recipes
Materials
E. coli KS272 (pKOBEG) grow on LB Cm (25 g/ml) at 30 C
The strain carries the pKOBEG plasmid (CmR) necessary for the recombination event and is used for generating recombined cosmids. The plasmid is compatible with ColE1-derived replicons. Expression of the red genes is induced by addition of 0.02-0.2 % arabinose. The plasmid pKOBEG is temperature sensitive so that all steps must be carried out at 30 C.

E. coli BW19610 (pCDA21) grow on LB Amp (50 g/ml) at 37 C
This strain carries a bi-functional selectable marker the Zeocin resistance gene (ZeoR) and the A. fumigatus pyrG gene (pyrG). Other cassettes may be available (e.g., using the E. coli hph gene, conferring hygromycin B resistance on A. fumigatus) but were not used here. The plasmid pCDA21 is used as the template for a PCR reaction.

A. fumigatus CEA17 or other pyrG deletion strain
In this strain pyrG can be used as a selectable marker. If different selectable markers are used (e.g., the hph gene) then a wild-type strain can also be transformed.

Strains and plasmids needed for this protocol can be obtained upon request from Dr. Christophe dEnfert, Unit Microbiologie et Environnement, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France. (Tel. +33 1 40 61 32 57, fax. +33 1 45 68 89 38, e-mail: denfert@pasteur.fr).

ZeocinTM can be obtained from e.g., Cayla, Toulouse, France (www.cayla.com), or Invitrogen, Groningen, The Netherlands (www.invitrogen.com).

LBLS LB with low salt concentration (5 % NaCl, 5 % yeast extract, 10 % peptone) since Zeocin is only effective at low salt concentrations.

A genomic cosmid library of A. fumigatus carrying the hygromycin B resistance marker was used (Langfelder et al., 1998).

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A rapid method for generating gene deletions in Aspergillus