Team:Cornell/project/wetlab/protoplasting

From 2013.igem.org

(Difference between revisions)
 
(11 intermediate revisions not shown)
Line 14: Line 14:
</div>
</div>
</div>
</div>
-
<div class="row last-ele">
+
<div class="row">
-
<div class="twelve columns">
+
<div class="nine columns">
-
<div class="nine columns">
+
<h3>Background</h3>
-
<h3>Background</h3>
+
Transformation of plant and fungal cells is difficult due to their cell walls that block passage of foreign DNA. Protoplasting is the method by which the cells walls of plant and fungal cells are digested to produce cells without cell walls, called protoplasts. The protoplasts can then be isolated from canidia by filtering through 20um nylon filter. Through additional methods, such as electroporation and PEG transformation, DNA can be uptaken by the protoplasts and then regrown into cells containing specific genes [1].  
-
Transformation of plant and fungal cells is difficult due to their cell walls that block passage of foreign DNA. Protoplasting is the method by which the cells walls of plant and fungal cells are digested to produce cells without cell walls, called protoplasts. Through additional methods, such as electroporation and PEG transformation, DNA can be uptaken by the protoplasts and then regrown into cells containing specific genes [1].  
+
</div>
-
 
+
                                 <div class="three columns center" style="font-size:10px; margin-top:-10px;">
-
                                </div>
+
-
                                 <div class="three columns center" style="font-size:10px; margin-top:-30px;">
+
<img src="https://static.igem.org/mediawiki/2013/f/fb/Protoplasts.jpg">
<img src="https://static.igem.org/mediawiki/2013/f/fb/Protoplasts.jpg">
-
                                        <i>Photo: Gillian Turgeon, Cornell University</i>
+
</div>
-
</div>
+
<div class="three columns center" style="font-size:10px; margin-top:-10px;">
-
+
<img src ="https://static.igem.org/mediawiki/2013/b/b0/CocheC5.jpg" style="margin-bottom:0px">
-
</div>
+
<br><i>Photo: Gillian Turgeon, Cornell University.</i>
-
+
-
<div class="twelve columns">
+
-
<div class="three columns">
+
-
<img src ="https://static.igem.org/mediawiki/2013/b/b0/CocheC5.jpg">
+
-
<i><font size="1">Photo: Gillian Turgeon, Cornell University.</font></i>
+
-
</div>
+
-
<div class="nine columns">
+
-
<h3>Methods</h3>Usually <i>Ganoderma lucidum</i> is protoplasted using lywallzyme and novozyme. However, these enzymes are exclusively available in China. Due to this, protoplasting was attempted with driselease and glucanex instead. After several attempts, we found that the enzymes are unable to digest the cell wall without also killing the cell. In addition, the <i>Ganoderma</i> mycelium was hard to pellet when centrifuging and made the protoplasting procedure difficult. Thus, <i>Cochliobolus heterostrophus</i> was used instead. Protoplasting was successful using driselease and glucanex and then transformed via PEG solution [2]. 
+
-
</div> 
+
</div>
</div>
 +
<div class="nine columns">
 +
<h3>Methods</h3>
 +
Usually <i>Ganoderma lucidum</i> is protoplasted using lywallzyme and novozyme. However, these enzymes are exclusively available in China. Due to this, protoplasting was attempted with driselease and glucanex instead. After several attempts, we found that the enzymes are unable to digest the cell wall without also killing the cell. In addition, the <i>Ganoderma</i> mycelium was hard to pellet when centrifuging and made the protoplasting procedure difficult. Thus, <i>Cochliobolus heterostrophus</i> was used instead. Protoplasting was successful using driselease and glucanex and then transformed via PEG solution [2]. 
 +
</div> 
 +
</div>
 +
<div class="row last-ele">
<div class="twelve columns">
<div class="twelve columns">
<h3>References</h3>
<h3>References</h3>

Latest revision as of 03:33, 29 October 2013

Cornell University Genetically Engineered Machines

Protoplasting

Background

Transformation of plant and fungal cells is difficult due to their cell walls that block passage of foreign DNA. Protoplasting is the method by which the cells walls of plant and fungal cells are digested to produce cells without cell walls, called protoplasts. The protoplasts can then be isolated from canidia by filtering through 20um nylon filter. Through additional methods, such as electroporation and PEG transformation, DNA can be uptaken by the protoplasts and then regrown into cells containing specific genes [1].

Photo: Gillian Turgeon, Cornell University.

Methods

Usually Ganoderma lucidum is protoplasted using lywallzyme and novozyme. However, these enzymes are exclusively available in China. Due to this, protoplasting was attempted with driselease and glucanex instead. After several attempts, we found that the enzymes are unable to digest the cell wall without also killing the cell. In addition, the Ganoderma mycelium was hard to pellet when centrifuging and made the protoplasting procedure difficult. Thus, Cochliobolus heterostrophus was used instead. Protoplasting was successful using driselease and glucanex and then transformed via PEG solution [2].

References

1. Sun L, Cai H, Xu W, Hu Y, Gao Y, Lin Z (2001). Efficient Transformation of the Medicinal Mushroom Ganoderma lucidum. Plant Molecular Biology Reporter, 19, 383a-383j.

2. Amir Sharon (ed.), Molecular and Cell Biology Methods for Fungi, Methods in Molecular Biology, vol. 638. Turgeon BG, Condon B, Liu J, Zhang N (2010). Protoplast Transformation of Filamentous Fungi.