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Team:Cornell/project/wetlab/fungal toolkit/fungal transformation
From 2013.igem.org
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<h2 class="centered">Fungal Transformation</h2> | <h2 class="centered">Fungal Transformation</h2> | ||
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</div> | </div> | ||
<div class="row"> | <div class="row"> | ||
| - | <div class="twelve columns"> | + | <div class="twelve columns"> |
| + | <h3>PEG Mediated Transformation</h3> | ||
| + | <h5>Background</h5> | ||
| + | Introduction of naked DNA into protoplasts gives opportunity for homologous recombination and random insertion for genomic integration. PEG mediated | ||
| + | transformation is a method by which naked DNA can be rapidly taken up by fresh protoplasts. Compared to other transformation methods including fungal | ||
| + | electroporation and agrobacterium mediated transformation, PEG mediated transformation is simple and yields a cell population with high survival and division | ||
| + | rates[1].<br> | ||
| + | <h5>Methods</h5> | ||
| + | In order to transform fungal cells via PEG mediated transformation, young mycelium must be grown in a liquid culture and subsequently protoplasted. By | ||
| + | exposing protoplasts to polyethylene glycol (PEG), DNA is rapidly uptaken by the cells giving the opportunity for random insertion or homologous recombination | ||
| + | for integration into the genome. It is hypothesized that PEG operates by binding to DNA neutralizing its negative charge. Through this, the cell is able to | ||
| + | uptake naked DNA. | ||
| + | <br> | ||
| + | <h5 style="margin-bottom:-20px">Transformants</h5> | ||
| + | <div class="four columns"> | ||
| + | <center><img src = "https://static.igem.org/mediawiki/2013/0/09/Linear_plasmid_2.jpg"></center> | ||
| + | <div style="font-size:11px; margin-top:-25px">Transformants of vector pHPVEL1, which conferred resistance against hygromycin.</div> | ||
| + | </div> | ||
| + | <div class="four columns"> | ||
| + | <center><img src = "https://static.igem.org/mediawiki/2013/0/04/DSC02398.JPG"></center> | ||
| + | <div style="font-size:11px; margin-top:-25px">Transformants of vector pNG, which conferred resistance against geniticin.</div> | ||
| + | </div> | ||
| + | <div class="four columns"> | ||
| + | <center><img src = "https://static.igem.org/mediawiki/2013/1/13/DSC02400.JPG"></center> | ||
| + | <div style="font-size:11px; margin-top:-25px">Transformants of a construct with GFP under the control of the trpc constitutive promoter between homology regions extracted from pNG.</div> | ||
| + | </div> | ||
| + | <div class="twelve columns"> | ||
| + | <center><img src = "https://static.igem.org/mediawiki/2013/6/60/Cochliobolus_fluorescence_composite.png" style="max-height:none"></center> | ||
| + | <div style="font-size:11px; margin-top:-25px">Fluorescence microscopy showed evidence of GFP expression in fungal mycelium transformed with a construct containing GFP under the control of the trpc promoter flanked by homology regions of <i>Cochliobolus heterostrophus</i> GPD. Wild type strains showed no signs of innate fluoresence.</div> | ||
| + | <br><br> | ||
| + | </div> | ||
| + | <h3>Agrobacterium Mediated Transformation</h3> | ||
| + | <h5>Background</h5> | ||
| + | Wide varieties of plant and fungal cells can be easily and efficiently transformed by agrobacterium-mediated transformation. In this process, plant or fungal | ||
| + | tissue is co-cultured with <i>Agrobacterium tumefaciens</i>, an organism that is able to transfer part of its DNA to plants and fungi, known as T-DNA. | ||
| + | Genetic constructs can be inserted into these T-DNA vectors and then transformed into <i>A. tumefaciens</i>, allowing it to insert this construct into a | ||
| + | plant or fungal genome [2]. | ||
| + | <br> | ||
| + | <center><img src = "https://static.igem.org/mediawiki/2013/9/90/Agrobacterium.png" style="max-height:none"></center> | ||
| + | <h5>Methods</h5> | ||
| + | In order to transform our genetic constructs into <i>Ganoderma lucidum</i>, we first inserted them into pOSCAR, a T-DNA vector that contains regions that <i> | ||
| + | A. tumefaciens</i> can insert into fungal cells. Following transformation of <i>A. tumefaciens</i> with our constructs in the pOSCAR vector, we co-cultured | ||
| + | the cells with <i>G. lucidum</i> in induction media that provides optimal conditions for T-DNA insertion for a period of 2 days. <i>G. lucidum</i> was then | ||
| + | placed on a selective plate to isolate transformants [3]. | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="twelve columns"> | <div class="twelve columns"> | ||
<h3>References</h3> | <h3>References</h3> | ||
| - | + | 1. Mathur, J., & Koncz, C. (1998). PEG-mediated protoplast transformation with naked DNA. In Arabidopsis Protocols (pp. 267-276). Humana Press. | |
| + | <br><br> | ||
| + | 2. Michielse, Caroline B., Paul J. J. Hooykaas, Cees A. M. J. J. Hondel, and Arthur F. J. Ram. "Agrobacterium-mediated Transformation as a Tool for | ||
| + | Functional Genomics in Fungi." Current Genetics 48.1 (2005): 1-17. | ||
| + | <br><br> | ||
| + | 3. Paz, Zahi, María D. García-Pedrajas, David L. Andrews, Steven J. Klosterman, Lourdes Baeza-Montañez, and Scott E. Gold. "One Step Construction of | ||
| + | Agrobacterium-Recombination-ready-plasmids (OSCAR), an Efficient and Robust Tool for ATMT Based Gene Deletion Construction in Fungi." Fungal Genetics and | ||
| + | Biology (2011): 677-684. | ||
</div> | </div> | ||
</div> | </div> | ||
Latest revision as of 03:43, 29 October 2013
Fungal Transformation
PEG Mediated Transformation
Background
Introduction of naked DNA into protoplasts gives opportunity for homologous recombination and random insertion for genomic integration. PEG mediated transformation is a method by which naked DNA can be rapidly taken up by fresh protoplasts. Compared to other transformation methods including fungal electroporation and agrobacterium mediated transformation, PEG mediated transformation is simple and yields a cell population with high survival and division rates[1].Methods
In order to transform fungal cells via PEG mediated transformation, young mycelium must be grown in a liquid culture and subsequently protoplasted. By exposing protoplasts to polyethylene glycol (PEG), DNA is rapidly uptaken by the cells giving the opportunity for random insertion or homologous recombination for integration into the genome. It is hypothesized that PEG operates by binding to DNA neutralizing its negative charge. Through this, the cell is able to uptake naked DNA.Transformants
Transformants of vector pHPVEL1, which conferred resistance against hygromycin.
Transformants of vector pNG, which conferred resistance against geniticin.
Transformants of a construct with GFP under the control of the trpc constitutive promoter between homology regions extracted from pNG.
Fluorescence microscopy showed evidence of GFP expression in fungal mycelium transformed with a construct containing GFP under the control of the trpc promoter flanked by homology regions of Cochliobolus heterostrophus GPD. Wild type strains showed no signs of innate fluoresence.
Agrobacterium Mediated Transformation
Background
Wide varieties of plant and fungal cells can be easily and efficiently transformed by agrobacterium-mediated transformation. In this process, plant or fungal tissue is co-cultured with Agrobacterium tumefaciens, an organism that is able to transfer part of its DNA to plants and fungi, known as T-DNA. Genetic constructs can be inserted into these T-DNA vectors and then transformed into A. tumefaciens, allowing it to insert this construct into a plant or fungal genome [2].
Methods
In order to transform our genetic constructs into Ganoderma lucidum, we first inserted them into pOSCAR, a T-DNA vector that contains regions that A. tumefaciens can insert into fungal cells. Following transformation of A. tumefaciens with our constructs in the pOSCAR vector, we co-cultured the cells with G. lucidum in induction media that provides optimal conditions for T-DNA insertion for a period of 2 days. G. lucidum was then placed on a selective plate to isolate transformants [3].References
1. Mathur, J., & Koncz, C. (1998). PEG-mediated protoplast transformation with naked DNA. In Arabidopsis Protocols (pp. 267-276). Humana Press.2. Michielse, Caroline B., Paul J. J. Hooykaas, Cees A. M. J. J. Hondel, and Arthur F. J. Ram. "Agrobacterium-mediated Transformation as a Tool for Functional Genomics in Fungi." Current Genetics 48.1 (2005): 1-17.
3. Paz, Zahi, María D. García-Pedrajas, David L. Andrews, Steven J. Klosterman, Lourdes Baeza-Montañez, and Scott E. Gold. "One Step Construction of Agrobacterium-Recombination-ready-plasmids (OSCAR), an Efficient and Robust Tool for ATMT Based Gene Deletion Construction in Fungi." Fungal Genetics and Biology (2011): 677-684.
