Team:NEAU-Harbin/Sandbox

From 2014.igem.org

(Difference between revisions)
Line 1: Line 1:
{{CSS/Main}}
{{CSS/Main}}
-
<!DOCTYPE html>
+
<!--DOCTYPE html-->
<html>
<html>
<head>
<head>
Line 1,044: Line 1,044:
border-right-style: inset;
border-right-style: inset;
}
}
-
/*等级积分*/
+
-
#s_info {
+
-
padding: 0px 0 0 0px;
+
-
color: #FFF;
+
-
border-top: 0px dotted #ccc;
+
-
margin-top: 8px;
+
-
text-align: center;
+
-
}
+
-
/*导航栏*/
+
-
.num {
+
-
left: 0px;
+
-
top: 394px;
+
-
background-color: #FFF;
+
-
width: 100%;
+
-
height: 40px;
+
-
z-index: 2;
+
-
float: right;
+
-
position: absolute;
+
-
right: 0px;
+
-
text-align: center;
+
-
padding-left: 225px;
+
-
color: #999;
+
-
padding-top: 5px;
+
-
}
+
-
/*导航栏-链接*/
+
-
.num a {
+
-
font-family: "Microsoft YaHei Light",微软雅黑 Light,"Microsoft JhengHei", "helvetica neue", Arial,"Lucida Grande", Sans-serif;
+
-
float: left;
+
-
display: block;
+
-
overflow: hidden;
+
-
font-size: 14px;
+
-
margin-right: 0px;
+
-
margin-left: 55px;
+
-
padding-top: 6px;
+
-
padding-right: 0px;
+
-
padding-bottom: 11px;
+
-
padding-left: 0px;
+
-
color: #999;
+
-
border-bottom-width: 2px;
+
-
border-bottom-style: solid;
+
-
border-bottom-color: #FFF;
+
-
/*动画*/
+
-
transition: 0.2s;
+
-
-moz-transition: 0.2s;
+
-
-webkit-transition: 0.2s;
+
-
-o-transition: 0.2s;
+
-
}
+
-
/*导航栏-链接-鼠标经过*/
+
-
.num a:hover {
+
-
border-top-width: 0px;
+
-
border-right-width: 0px;
+
-
border-bottom-width: 2px;
+
-
border-left-width: 0px;
+
-
border-top-style: solid;
+
-
border-right-style: solid;
+
-
border-bottom-style: solid;
+
-
border-left-style: solid;
+
-
border-top-color: #FFF;
+
-
border-right-color: #FFF;
+
-
border-bottom-color: #06F;
+
-
border-left-color: #FFF;
+
-
color: #666;
+
-
}
+
-
/*导航栏-链接-激活状态*/
+
-
.num a.on {
+
-
-moz-border-radius: 0px 0px 0 0;
+
-
-khtml-border-radius: 0px 0px 0 0;
+
-
-webkit-border-radius: 0px 0px 0 0;
+
-
background-image: none;
+
-
background-repeat: repeat-x;
+
-
background-position: 0 0px;
+
-
border-top-width: 0px;
+
-
border-right-width: 0px;
+
-
border-bottom-width: 2px;
+
-
border-left-width: 0px;
+
-
border-top-style: solid;
+
-
border-right-style: solid;
+
-
border-bottom-style: solid;
+
-
border-left-style: solid;
+
-
border-top-color: #FFF;
+
-
border-right-color: #FFF;
+
-
border-bottom-color: #06F;
+
-
border-left-color: #FFF;
+
-
color: #333;
+
-
font-weight: bolder;
+
-
}
+
</style>
</style>
<!--transpicbox end-->
<!--transpicbox end-->
Line 1,494: Line 1,409:
<!--requirements section -->
<!--requirements section -->
-
<tr><td colspan="3"> <h3> Requirements </h3></td></tr>
+
<tr><td colspan="3"> <h3> Description </h3></td></tr>
<tr>
<tr>
<td width="45%"  valign="top">  
<td width="45%"  valign="top">  
-
<ol>Target</ol><p>
+
<p>
-
Firstly, we aim to introduce the new expression host Aspergillus Niger, which is a significant industrial fermentation microbial to iGEM. It's well known for efficient capacity of secreting proteins and recognized as the safe strain for heterologous protein production. Secondly, we want to exploit the visual gene replacement system. We design to utilize the fluorescent proteins to realize the indication of the target gene replacement and make the process continuable. The fluorescent proteins work as selective markers to help us pick out our target transformants without complex molecular detections. Thirdly, we import carotenoid metabolic pathway where all the enzymes are connected by the linker peptides to our vector, so that we can examine the feasibility of our system.
+
<i>Aspergillus Niger</i> is one species of filamentous fungi which have been widely employed in the fermentation industry, and become a principal source of enzymes and metabolites. With features such as low cost and high productivity and Generally Regarded As Safe (GRAS) status in the food and food processing industry, <i>Aspergillus Niger</i> has achieved increased attention as host for the production of heterologous proteins, such as amylase, acid protease, cellulase, pectinase and glucose oxidase, etc.</p><p>
-
 
+
In this study, we aim to establish one visual operation system of continuous target gene replacement using <i>Aspergillus Niger</i>. Firstly, a construct contains amilCP gene, which produces blue chromoproteins, and cjBlue, which produces green chromoproteins, fused selective marker gene HPH was made and transformed into the high expression site through homologous recombination in the <i>Aspergillus Niger</i> genome. In this construct, amilCP gene was driven by GlaA5 promoter, and followed by GlaA3 terminator; the HPH and cjBlue fusion gene was driven by pgpdA promoter, and followed by GlaA3 terminator. GlaA5 and GlaA3 can be used as homologous arm sequences that can mediate specific recombination. The selected HPH resistant <i>Aspergillus Niger</i> cell will highly express amilCP and cjBlue protein which can be detected by <reds>blue color</reds> and cjBlue signal. After several generations, homologous recombination will happen between the two GlaA3 sequences, and lead to the loss of HPH and cjBlue, then get the transgenic <i>Aspergillus Niger</i> cells only express amilCP gene which can be easily detected by <reds>blue color</reds>. These transgenic <i>Aspergillus Niger</i> cells with <reds>blue color</reds> can be used as original strain for target gene transformation. Those colonies without <reds>blue color</reds> will be the real transformants in which amilCP gene is replaced with the target gene. In a word, the visual gene operation system in <i>Aspergillus Niger</i> will be established with which the homologous recombination of target genes will be easily traced by detecting the color of colonies.
-
 
+
</p><p>So far we have finished the expression vectors construction. The genetic transformation work is in the process.
-
Now we are still constructing our expression vectors. We have ligated HPH, GFP with linker and one of GlaA3s. Now we are trying to ligate GlaA5 and another GlaA3. When we finish our expression vector, we will transform the well-constructed plasmid into the Agrobacterium and use the Agrobacterium to infect the Aspergillus niger to carry our plasmid in the Aspergillus niger. </p>
+
</p>
-
 
+
 +
<br /><br /><br />
</body>
</body>
</html>
</html>

Revision as of 13:06, 14 August 2014

Welcome to my iGEM
Hello world!



Thanks for visiting our wiki!

Our team has been approved and we are ready to start the iGEM season!
On this page we can document our project, introduce our team members, document our progress
and share our iGEM experience with the rest of the world!


Click here to edit this page!

Description

Aspergillus Niger is one species of filamentous fungi which have been widely employed in the fermentation industry, and become a principal source of enzymes and metabolites. With features such as low cost and high productivity and Generally Regarded As Safe (GRAS) status in the food and food processing industry, Aspergillus Niger has achieved increased attention as host for the production of heterologous proteins, such as amylase, acid protease, cellulase, pectinase and glucose oxidase, etc.

In this study, we aim to establish one visual operation system of continuous target gene replacement using Aspergillus Niger. Firstly, a construct contains amilCP gene, which produces blue chromoproteins, and cjBlue, which produces green chromoproteins, fused selective marker gene HPH was made and transformed into the high expression site through homologous recombination in the Aspergillus Niger genome. In this construct, amilCP gene was driven by GlaA5 promoter, and followed by GlaA3 terminator; the HPH and cjBlue fusion gene was driven by pgpdA promoter, and followed by GlaA3 terminator. GlaA5 and GlaA3 can be used as homologous arm sequences that can mediate specific recombination. The selected HPH resistant Aspergillus Niger cell will highly express amilCP and cjBlue protein which can be detected by blue color and cjBlue signal. After several generations, homologous recombination will happen between the two GlaA3 sequences, and lead to the loss of HPH and cjBlue, then get the transgenic Aspergillus Niger cells only express amilCP gene which can be easily detected by blue color. These transgenic Aspergillus Niger cells with blue color can be used as original strain for target gene transformation. Those colonies without blue color will be the real transformants in which amilCP gene is replaced with the target gene. In a word, the visual gene operation system in Aspergillus Niger will be established with which the homologous recombination of target genes will be easily traced by detecting the color of colonies.

So far we have finished the expression vectors construction. The genetic transformation work is in the process.




Retrieved from "http://2014.igem.org/Team:NEAU-Harbin/Sandbox"