Navigation Links
Transformation of Filamentous Fungi by High-Voltage Electroporation

Contributed by G.H. Goldman, R. Geremia, M. Van Montagu, and A. Herrera-Estrella, Laboratorium voor Genetica, Rijksuniversiteit Gent, B-9000 Gent, Belgium


Introduction
Virtually all fungal transformation protocols call for the addition of a high concentration of polyethylene glycol (PEG) following the initial period of exposure to DNA. However, electroporation could also be useful for introducing genes into filamentous fungi. Recently, electroporation has become a valuable technique for transferring nucleic acids into both eukaryotic and prokaryotic cells (Miller et al., 1988; Fster and Neumann, 1989). In this report, we show the use of high voltage-mediated transformation as an efficient method for genetic transformation of Trichoderma harzianum with plasmid DNA.


Materials and Methods
Strains and plasmid. T. harzianum strain IMI206040 was used as the transformation host. The plasmid used was pAN7-1, a derivative of pUC19 containing the E. coli hygromycin B phosphotransferase gene as a dominant selectable marker, and the gpd promoter and trpC terminator signals from Aspergillus nidulans (Punt et al., 1987). Plasmid DNA was purified from E. coli MC1061 by standard procedures (Maniatis et al., 1982) and dialyzed against distilled water prior to use.

Electro-competent cells. Osmotically sensitive cells (OSC) were prepared according to Laurila et al. (1985) with the following modifications: cellophane sheets placed on Potato Dextrose Agar (PDA) plates were inoculated with 5 x 106 spores/ml and incubated for 21 hours at 28 C; the germinative tubes from five cellophane sheets were suspended in 15 ml of buffer (1.2 M MgSO4, 10 mM sodium phosphate, pH 5.8) containing Novozyme-234 (5 mg/ml) in a Petri dish; and, these plates were incubated at 28 C for 30 minutes with agitation in a rotary shaker at 150 rpm. The OSC generated with this treatment were then centrifuged in corex tubes at 4,000 x g for 5 minutes and the pellet was washed twice with 1.2 M sorbitol in water. After the last wash, the OSC were resuspended in 1.2 M sorbitol at the desired concentration.

Transformation by electroporation. Two micrograms of transforming DNA were mixed with 400 l of the OSC suspension and kept on ice. High voltage pulses were delivered to 400 l samples in 0.2 cm electrode gap cuvettes (Bio-Rad Laboratories) by using a Gene Pulser apparatus with the Pulse Controller (Bio-Rad Laboratories). Following delivery of the electrical pulse, OSC were mixed with 1.0 ml of Potato Dextrose Broth (PDB) plus 1.2 M sorbitol (PDBS), incubated for 10 minutes on ice, and then for 2 hours at 28 C. When PEG was used, the following modifications were made: before adding the transforming DNA, spheroplasts were centrifuged at 4,000 x g for 15 minutes; the pellet was resuspended in 400 l of 1.2 M sorbitol plus 1.0% PEG 6000 (Fluka); and, after electroporation, the OSC were mixed with 5.0 ml PDBS. After the incubation period of either treatment, aliquots were plated using an agar overlay on plates containing PDA plus 1.2M sorbitol and 100 g/ml of hygromycin B as previously described by Herrera-Estrella et al. (1990).

DNA preparation. DNA was isolated from T. harzianum mycelia grown in liquid cultures in PDB medium plus 20 g/ml of hygromycin B (Calbiochem) according to the method described by Raeder and Broda (1985).


Results
Electroporation of germinative tubes or mycelia from T. harzianum did not yield transformants, so osmotically sensitive cells were tested. The effect of incubation time of the germinative tubes with the cell wall-degrading enzymes on the electro-transformation efficiency was tested. Intervals of 15, 30, and 45 minutes were used. An incubation period shorter than 30 minutes did not yield any transformants while a 45 minute incubation yielded 50% less transformants than did a 30 minute incubation time (data not shown). To optimize conditions, different combinations of voltages and capacitances were chosen, while holding the parallel resistor at 200 ohms and holding the concentration of OSC at 2.0 x 108 per ml (data not shown). The electroporation medium consisted of distilled water with 1.2 M sorbitol as an osmotic protectant. These preliminary results indicated that transformation could be obtained at 2.0 kV/cm with a capacitance of 25 F. Figure 1A shows that this was a good approximation since the maximum yield of transformants was found at 2.8 kV/cm.

The effect of the parallel resistor (and thus the time constant, τ=RxC) on electroporation efficiency was also examined (Figure 1B). The best yield was obtained when the pulse was delivered using a field strength of 2.0 kV/cm, a parallel resistance of 800 ohms and a capacitance of 25 F. Using 2.0 kV/cm with 25 F, the survival of the OSC decreased sharply at 100 ohms reaching 20% survival, and continued to decrease slowly from 200 ohms to 800 ohms. At 800 ohms, the survival was about 10.0%. From these initial experiments, the best electrical conditions found for the electroporation of T. harzianum were a field strength of 2.8 kV/cm with a capacitance of 25 F and a parallel resistance of 800 ohms. T. harzianum OSC subjected to these conditions in the absence of the pAN7-1 plasmid did not produce any hygromycin-resistant colonies.

Total cellular DNA of several independent transformants was isolated. Undigested DNA, as well as DNA digested with EcoRV, BamHI, HindIII, and EcoRI was subjected to agarose gel electrophoresis and analyzed by Southern blot using pAN7-1 as a probe. Wild-type T. harzianum contained no sequences hybridizing to the vector. Figure 2 shows the pattern of hybridization for three transformants. The pAN7-1 plasmid is about 6.5 kb in size, and it has no EcoRV sites, one site each for BamHI and HindIII, and two sites for EcoRI (which yields two fragments of about 3.9 and 2.6 kb). The digestions showed the restriction patterns expected from the restriction map of pAN7-1. Each transformant most likely contains tandem repeats of the vector. Hybridization of undigested DNA occurred only in the high molecular weight genomic band (UC), suggesting that pAN7-1 integrated into the genome and did not replicate autonomously.

The effect of the number of electro-competent cells on the transformation efficiency has already been observed in bacteria, mammalian cells, and plant protoplasts (Fromm et al., 1985; Dower et al., 1988; Miller et al., 1988; Shigekawa and Dower, 1988). We investigated the effect of the number of electro-competent T. harzianum cells on the transformation efficiency. By increasing the concentration of OSC to about 1.0 x 109/ml, we were able to increase the transformation efficiency 2.3-fold. When a concentration of 2.5 x 109 OSC/ml was used, a sharp decrease in the number of transformants/g of DNA occurred. This low efficiency correlated with a decrease in the time constant probably caused by the high concentrations of OSC providing a higher resistance (data not shown). A sharp decrease in the transformation efficiency was observed when concentrations of OSC lower than 2.0 x 108/ml were used. The effect of plasmid DNA concentration on the number of transformants obtained by the electroporation of identical quantities of cells was also examined. The transformation efficiency increased with increasing concentrations (up to 40 g/ml) of plasmid DNA (data not shown).

To date, all protocols known for the chemical transformation of filamentous fungi are based on the utilization of PEG. Figure 3 shows the influence of different concentrations of PEG on the transformation efficiency. The use of 1.0% PEG in the electroporation medium made it possible to obtain an efficiency of 435 transformants/g of DNA. This efficiency is about four times greater than electroporation without PEG. A control pulse using only PEG in the same concentration did not yield any transformants. Recently, efficient transformation of Rhodococcus fascians (Desomer et al., 1990) and Bacillus thuringiensis (Mahillon et al., 1989) has been obtained by combining PEG and electroporation.

The decreased efficiency of transformation obtained by increasing the concentration of PEG above 1% did not correlate to the survival of the OSC (data not shown). Unexpectedly, the number of transformants was lower when concentrations of PEG below 1% were used than in the control treatment without PEG. This behavior was found to be reproducible in at least three independent experiments. Volumes smaller than 400 l produced lower numbers of transformants. This result could be expected since reducing the cross-sectional area of the solution at the electrode surface increases the resistance (Shigekawa and Dower, 1988).


Conclusions
There are two major advantages of electroporation over the traditional chemical method of transformation. The first is simplicity: the OSC do not need to be purified by sorbitol gradients, and it is possible to perform many electro-transformations at one time. The second advantage we found is that electroporation is more reproducible than PEG-mediated transformation. An important difference we observed between the two methods was the stability of transformants from T. harzianum when transformed with the plasmid pAN7-1. A possible explanation for this effect could be the activation of the repair system caused by the high-voltage electric pulse.

The results presented here show that OSC from T. harzianum can be efficiently transformed by electroporation. Electroporation is rapid, easy to perform, and requires minimal sample preparation. Therefore, it may prove to be a general method useful for introducing DNA into many fungal species in addition to T. harzianum, A. nidulans (Richey et al., 1989), F. solani (Richey et al., 1989), S. cerevisiae (Delorme, 1989; Hashimoto et al., 1985; Hill, 1989; Karube et al., 1985; Meilhoc et al., 1990; Rech et al., 1990; Weaver et al., 1988), S. pombe (Hood and Stachow, 1990; Weaver et al., 1988), and D. discoideum (Dynes and Firtel, 1989; Egelhoff et al., 1989; Howard et al., 1988). This report presents possibilities to improve transformation systems that have already been described, or to transform other filamentous fungi where PEG-mediated transformation has not been achieved.


References
Delorme, E., Appl. Environ. Microbiol., 55, 2242 (1989).

Desomer, J., Dhaese, P., and Van Montagu, M., Appl. Environ. Microbiol., 56, 2818 (1990).

Dower, W. J., Miller, J. F., and Ragsdale, C. W., Nucl. Acids Res., 16, 6127 (1988).

Dynes, J. L. and Firtel, R. A., Proc. Natl. Acad. Sci. USA, 86, 7966 (1989).

Egelhoff, T. T., Brown, S. S., Manstein, D. J., and Spudich, J. A., Molec. Cell. Biol., 9, 1965 (1989).

Fster, W. and Neumann, E., Gene Transfer by Electroporation. A Practical Guide. In Neumann, E., Sowers, E. A., and Jordan, C. A., (eds) Electroporation and Electrofusion in Cell Biology, Plenum Press, New York (1989).

Fromm, M., Taylor, L. P., and Walbot., V., Proc. Natl. Acad. Sci. USA, 82, 5824 (1985).

Hashimoto, H., Morikawa, H., Yamada, Y., and Kimura, A., Appl. Microbiol. Biotechnol., 21, 336 (1985).

Herrera-Estrella, A., Goldman, G. H., and Van Montagu, M., Molec. Microbiol., 4, 839 (1990).

Hill, D. E., Nucl. Acids Res., 17, 8011 (1989).

Hood, M. T. and Stachow, C., Nucl. Acids Res., 18, 688 (1990).

Howard, P. K., Ahern, K. G., and Firtel, R. A., Nucl. Acids Res., 16, 2613 (1988).

Karube, I., Tamiya, E., and Matsuoka, H., FEBS Lett., 182, 90 (1985).

Laurila, H. O., Nevalainen, H., and Mkinen, V., Appl. Microbiol. Biotechnol., 21, 210 (1985).

Mahillon, J., Chungjatupornchai, W., Decock, J. Dierickx, S., Michiels, F., Peferoen, M., and Joos, H., FEMS Microbiol. Lett., 60, 205 (1989).

Maniatis, T., Fritsch, E. F., and Sambrook, J., Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory, New York (1982).

Meilhoc, E., Masson, J.-M., and Teissi, J., Bio/Technol., 8, 223 (1990).

Miller, J. F., Dower, W. J., and Tompkins, L. S., Proc. Natl. Acad. Sci. USA, 85, 856 (1988).

Punt, P. J., Oliver, R. P., Dingemanse, M. A., Pouwels, P. H., and van den Hondel, C. A. M. J. J., Gene 56, 117 (1987).

Raeder, U. and Broda, P., Lett. Appl. Microbiol. 1, 17 (1985).

Rech, E. L., Dobson, M. J., Davey, M. R., and Mulligan, B. J., Nucl. Acids Res., 18, 1313 (1990).

Richey, M. G., Marek, E. T., Schardl, C. L., and Smith, D. A., Phytopathology, 79, 844 (1989).

Shigekawa, K. and Dower, W. J., BioTechniques, 6, 742 (1988).

Weaver, J. C., Harrison, G. I., Bliss, J. G., Mourant, J. R., and Powell, K. T., FEBS Lett., 229, 30 (1988).


back to top
'"/>

Source:


Page: All 1 2 3 4 5 6 7 8

Related biology technology :

1. Highest Possible Transformation Efficiencies for High-Throughput Applications
2. New Competent Cells for Highest Transformation Efficiencies
3. Optimization of Biolistic Transformation Using the Helium-Driven PDS-1000/He System
4. Sub-Micron Gold Particles Are Superior to Larger Particles for Efficient Biolistic Transformation of Organelles and Some Cells
5. Transformation of Nematodes With the Helios Gene Gun
6. Transformation of Filamentous Fungi by Microprojectile Bombardment
7. Eppendorf Electroporation Application Notes Your participation is highly welcome !!!
8. Eppendorf Electroporation Application Notes Your participation is highly welcome !!!
9. Efficient Delivery of siRNAs to Human Primary Cells: Electroporation vs. Chemical Transfection
10. Optimizing Chemical Transfection and Electroporation of siRNAs
11. High Throughput siRNA Electroporation
Post Your Comments:
(Date:6/30/2015)... , June 30, 2015  Juniper Pharmaceuticals, ... focused on developing therapeutics that address unmet medical ... George Elston will present at the ... Wednesday, July 8, 2015Time: , 4:45 PM EDTLocation: ... (live & archive): , www.juniperpharma.com, under  ,Investor, ...
(Date:6/30/2015)... ... ... The maximum number of shares proposed to be purchased in the tender offer ... stock). On June 29, 2015, the last trading day prior to the commencement of ... was $2.29 per share. , The tender offer will expire on July 28, 2015 ...
(Date:6/30/2015)... 30, 2015  The Dr. Samadi Prostate Cancer Center in ... a full suite of new genetic testing methods for men ... diagnosing prostate cancer. Dr. Samadi,s Prostate ... for assessing the risk and optimizing the diagnosis of prostate ... excited to now offer these revolutionary genetic tests to men ...
(Date:6/29/2015)... , June 30, 2015   Pharnext SAS today ... developed for the treatment of Charcot-Marie-Tooth Type 1A (CMT1A), is ... the 2015 Peripheral Nerve Society (PNS) Biennial Meeting at the ... , June 27 - July 2, 2015. At ... Wed July 1, 5p.m. to 7p.m. EDT: ...
Breaking Biology Technology:Juniper Pharmaceuticals to Present at Cantor Fitzgerald Inaugural Healthcare Conference on July 8, 2015 2Cryo-Cell Commences Tender Offer to Purchase its Common Stock, Up to 750,000 Shares at $3.25 per Share 2Cryo-Cell Commences Tender Offer to Purchase its Common Stock, Up to 750,000 Shares at $3.25 per Share 3New Revolutionary Genetic Testing for Prostate Cancer Offered at Dr. Samadi's Prostate Cancer Center 2New Revolutionary Genetic Testing for Prostate Cancer Offered at Dr. Samadi's Prostate Cancer Center 3New Revolutionary Genetic Testing for Prostate Cancer Offered at Dr. Samadi's Prostate Cancer Center 4New Revolutionary Genetic Testing for Prostate Cancer Offered at Dr. Samadi's Prostate Cancer Center 5Pharnext's PXT-3003 Featured in Multiple Presentations at 2015 Peripheral Nerve Society Biennial Meeting 2
... Top 25 Global Pharmaceutical Companies Select Drug ... Visualization, MOUNTAIN VIEW, Calif., April 2, 2008 ... provider of software,strategic consulting, and regulatory services for ... 25 global pharmaceutical,companies have become customers for its ...
... 1, 2008 MiMedx Group, Inc.,(OTC Bulletin Board: ... AYXC), today reported that the contemplated reverse stock ... related name change, to,MiMedx Group, Inc., was approved ... a result of the reverse split and redomestication, ...
... Installation and Major Data Migration in ... United States and Abroad, CHICAGO, April 1, ... signed an agreement with the Cleveland,Clinic, a not-for-profit ... deploy the syngo(R) Dynamics cardiology image,management and reporting ...
Cached Biology Technology:Pharsight Signs Two New DMX(R) License Customers 2Pharsight Signs Two New DMX(R) License Customers 3MiMedx Group Announces Post-split Name Change 2MiMedx Group Announces Post-split Name Change 3Siemens Signs Multi-Year Agreement With Cleveland Clinic to Deploy Enterprise-Wide Cardiology PACS 2
(Date:6/18/2015)... June 18, 2015 NXT-ID, Inc. (NASDAQ: NXTD ... on the growing mobile commerce market announces that its Wocket® ... on "Money on the Mark", scheduled to air on WABC ... June 20 th . The broadcast air- ... 7:00 to 8:00pm EST. NXT-ID, Inc.,s CEO Gino Pereira ...
(Date:6/16/2015)... , June 16, 2015 /CNW Telbec/ - ... partnership centralized around the incorporation of handyem,s HPC-150 ... diagnostic laboratory, the Mo-POD™. This unprecedented model of ... International Conference at the Pennsylvania ... from June 15 th to 18 th ...
(Date:6/15/2015)... , June 15, 2015 A ... from Telstra reveals the majority of US consumers using mobile ... via biometrics, such as fingerprint and voiceprint, instead of having ... According to Telstra,s " Mobile Identity   -   ... report, with smartphones now the primary channel used by ...
Breaking Biology News(10 mins):NXT-ID, Inc.'s CEO Gino Pereira Talks About the Wocket Smart Wallet on WABC Radio, New York City, June 20th 2NXT-ID, Inc.'s CEO Gino Pereira Talks About the Wocket Smart Wallet on WABC Radio, New York City, June 20th 3handyem and FlowMetric Diagnostics announce their partnership in the development of Flow Cytometry- Based Solutions in diagnostic capabilities 2handyem and FlowMetric Diagnostics announce their partnership in the development of Flow Cytometry- Based Solutions in diagnostic capabilities 3One in Four US Consumers Would Share Their DNA With Their Bank to Secure Financial and Personal Information, Telstra Report Finds 2One in Four US Consumers Would Share Their DNA With Their Bank to Secure Financial and Personal Information, Telstra Report Finds 3One in Four US Consumers Would Share Their DNA With Their Bank to Secure Financial and Personal Information, Telstra Report Finds 4
... , ,Saskatoon (November 3, 2008) We all know physical ... for you? What effect does exercise have on the cells and ... that we can use physical activity more effectively to combat chronic ... and psychological factors prevent people from exercising or playing sports? ...
... YORK (Nov. 6, 2008) -- On Nov. 8 ... and Columbia University Medical Center will host an ... Hyatt New York, which will feature lectures by ... findings and stem cell therapy breakthroughs. Key ...
... General Hospital (MGH) is giving what may be the ... key symptom of schizophrenia, impaired working memory. Functional ... in two genes is associated with reduced activity of ... normal controls. The report has been released online ...
Cached Biology News:Physical activity and health: Finding the right prescription 2Innovations in Pediatric Medicine International Conference brings together pediatrics experts 2Interaction between gene variants may alter brain function in schizophrenia 2
... This antibody is specific for human cardiac troponin ... the Ca-binding signals responsible for contraction of cardiac ... troponin complex in human cells. It also cross-reacts ... Antigen: Highly purified human ...
... N-(2-Naphthoyl)-Val-phenylalaninal 2-Naphthoyl-VF-CHO White ... INERT GAS. A cell-permeable, reversible inhibitor of ... x-40 (ED 50 = 2.6 ... = 2.7 μM) in HEK293 cells ...
cystatin A (N-18)...
...
Biology Products: