Team:Freiburg Bioware/NoteBook/Labjournal/April

From 2010.igem.org

(Difference between revisions)
(→Theoretical cloning)
 
(33 intermediate revisions not shown)
Line 1: Line 1:
 +
{{:Team:Freiburg_Bioware/css}}
 +
{{:Team:Freiburg_Bioware/Head}}
 +
{{:Team:Freiburg_Bioware/menu_notebook}}
 +
{{:Team:Freiburg_Bioware/jquery}}
 +
<!-- Freiburg_bioware -->
<!-- Freiburg_bioware -->
 +
[https://2010.igem.org/Team:Freiburg_Bioware/NoteBook => Back to Notebook overview]<br><br>
<html>
<html>
-
<!-- Stylesheed -->
+
<div class="box_right">
-
<style>
+
<left><u1>NoteBook Navigator</u1></left>
 +
<br>
-
#content {
+
<ul>
-
width: 965px;
+
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March">March (labday 1)</a></li>
-
/*background-color: #3300ee;*/
+
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April">April (labday 2 - 5)</a></li>
-
}
+
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May">May (labday 6 - 17)</a></li>
 +
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/June">June (labday 18 - 45)</a></li>
 +
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/July">July (labday 46 - 75)</a></li>
 +
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August">August part 1 (labday 76 - 92)</a></li>
 +
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August2">August part 2 (labday 93 - 106)</a></li>
 +
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li>
 +
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li>
 +
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 149 )</a></li>
 +
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 150 - 166 )</a></li>
 +
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November  (labday 167 - 170 )</a></li>
 +
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture</a></li>
 +
</ul>
 +
</div>
 +
</html>
-
#banner_top {
 
-
opacity: 1;
 
-
width: 965px;
 
-
height: 200px;
 
-
margin: 0px 0px 0px 0px;
 
-
}
 
-
/*Menu and the corresponding buttons*/
 
-
#menu {
+
===<p style="font-size:17px; background-color:#00dd77;">2. Labday 13.04.2010</p>===
-
width: 900px;
+
-
margin: 30px 0px 30px 0px;
+
-
}
+
-
 
+
-
div.menu_button {
+
-
float: left;
+
-
width: 110px;
+
-
}
+
-
 
+
-
div.menu_button a {
+
-
font-weight: bold;
+
-
font-size: 12px;
+
-
/*border: 1px solid black;*/
+
-
text-decoration: none;
+
-
color: #555555;
+
-
background: transparent url(http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/7/7e/Freiburg10_Buttongrau100.png) no-repeat scroll center center;
+
-
}
+
-
 
+
-
div.menu_button a:hover {
+
-
/*color: #999999;*/
+
-
background: transparent url(http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/9/99/Freiburg10_Buttongruen100.png) no-repeat scroll center center;
+
-
}
+
-
 
+
-
#BHome a {
+
-
padding: 20px 35px 20px 35px;
+
-
margin: 10px 2px 10px 2px;
+
-
}
+
-
#BTeam a {
+
-
padding: 20px 35px 20px 35px;
+
-
margin: 10px 2px 10px 2px;
+
-
}
+
-
#BProject a {
+
-
padding: 20px 30px 20px 30px;
+
-
margin: 10px 2px 10px 2px;
+
-
}
+
-
#BBioBricks a {
+
-
padding: 20px 15px 20px 15px;
+
-
margin: 10px 2px 10px 2px;
+
-
}
+
-
#BNote a {
+
-
padding: 20px 22px 20px 21px;
+
-
margin: 10px 2px 10px 2px;
+
-
}
+
-
#BSponsors a {
+
-
padding: 20px 24px 20px 22px;
+
-
margin: 10px 2px 10px 2px;
+
-
}
+
-
#BSafety a {
+
-
padding: 20px 32px 20px 32px;
+
-
margin: 10px 2px 10px 2px;
+
-
}
+
-
#BModelling a {
+
-
padding: 20px 24px 20px 24px;
+
-
margin: 10px 2px 10px 2px;
+
-
}
+
-
 
+
-
/* Main area */
+
-
 
+
-
div.overall {
+
-
width: 965px;
+
-
margin: 30px 0px 0px 0px;
+
-
}
+
-
 
+
-
#subMenu {
+
-
width: 165px;
+
-
height: 600px;
+
-
float: left;
+
-
margin: 40px 10px 0px 0px;
+
-
border: 1px solid black;
+
-
}
+
-
 
+
-
div.box_right {
+
-
width: 780px;
+
-
height: auto;
+
-
float: left;
+
-
margin: 40px 0px 0px 0px;
+
-
/*border: 1px solid black;*/
+
-
}
+
-
</style>
+
-
<!-- Start stuff you see -->
+
-
 
+
-
<!-- Banner -->
+
-
<img src="http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/3/31/Freiburg10_Header_wiki.png" alt="banner" id="banner_top"/>
+
-
 
+
-
<!-- Menu -->
+
-
<center>
+
-
<div id="menu">
+
-
<div class="menu_button" id="BHome"><a href="http://www.molbiotech.uni-freiburg.de/iGEM" target="blank">Home</a></div>
+
-
<div class="menu_button" id="BTeam"><a href="http://www.molbiotech.uni-freiburg.de/iGEM" target="blank">Team</a></div>
+
-
<div class="menu_button" id="BProject"><a href="http://www.molbiotech.uni-freiburg.de/iGEM" target="blank">Project</a></div>
+
-
<div class="menu_button" id="BBioBricks"><a href="http://www.molbiotech.uni-freiburg.de/iGEM" target="blank">BioBricks &trade;</a></div>
+
-
<div class="menu_button" id="BNote"><a href="http://www.molbiotech.uni-freiburg.de/iGEM" target="blank">Note Book</a></div>
+
-
<div class="menu_button" id="BSponsors"><a href="http://www.molbiotech.uni-freiburg.de/iGEM" target="blank">Sponsors</a></div>
+
-
<div class="menu_button" id="BSafety"><a href="http://www.molbiotech.uni-freiburg.de/iGEM" target="blank">Safety</a></div>
+
-
<div class="menu_button"id="BModelling"><a href="http://www.molbiotech.uni-freiburg.de/iGEM" target="blank">Modelling</a></div>
+
-
</div>
+
-
</center>
+
-
 
+
-
<!-- Main area -->
+
-
<div id="subMenu">
+
-
</div>
+
-
<div class="box_right">
+
-
<ul>
+
-
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March">March</a></li>
+
-
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April">April</a></li>
+
-
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May">May</a></li>
+
-
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/June">June</a></li>
+
-
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/July">July</a></li>
+
-
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August">August</a></li>
+
-
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September</a></li>
+
-
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October</a></li>
+
-
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November</a></li>
+
-
</ul>
+
-
 
+
-
</html>
+
-
===2. Labday 13.04.2010===
+
====<p style="font-size:15px; background-color:#66bbFF;"><b>The Biobricks Foundation: RFC</b></p>====
====<p style="font-size:15px; background-color:#66bbFF;"><b>The Biobricks Foundation: RFC</b></p>====
<p><b>Investigators: Chris W., Bea, Kira, Hanna, Julian, Anna, Jessica (Tobias,Sven, Kristian)</b></p><br>
<p><b>Investigators: Chris W., Bea, Kira, Hanna, Julian, Anna, Jessica (Tobias,Sven, Kristian)</b></p><br>
-
 
+
The BioBricks Foundation is dedicated to promoting and protecting open development, sharing, and reuse of BioBrick™ standard biological parts.<br>
-
 
+
-
The BioBricks Foundation is dedicated to promoting and protecting the open development, <br>
+
-
sharing, and reuse of BioBrick™ standard biological parts.<br>
+
Homepage: [[http://openwetware.org/wiki/The_BioBricks_Foundation:RFC]]
Homepage: [[http://openwetware.org/wiki/The_BioBricks_Foundation:RFC]]
-
Two important standarts:<br>
+
Two important standards:<br>
'''BBF RFC-10''':<br>
'''BBF RFC-10''':<br>
Line 161: Line 55:
*Thymidin Kinase HSVI <br>  
*Thymidin Kinase HSVI <br>  
Accesion: V00470 (Genebank)
Accesion: V00470 (Genebank)
-
CDS without iGEM-restriction site, unkown sequence in front of CDS with an EcoRI-restriction site, sequence from 1980! (there are a lot of X-ray analysis, but just to be on the safe side we should check the sequence)
+
CDS without iGEM-restriction sites, unkown sequence in front of CDS with an EcoRI-restriction site, sequence from 1980! (There is a lot of X-ray crystallography analysis data available, but just to be on the safe side we should check the sequence again)
*Cytosine Deaminase (see also FCY1) ('''S.cerevisae''')<br>  
*Cytosine Deaminase (see also FCY1) ('''S.cerevisae''')<br>  
Line 171: Line 65:
*Alignment of the amino acid-sequences of the cytosin deaminases of E.coli (Invivogen) and S.cerevisiae (Genebank)
*Alignment of the amino acid-sequences of the cytosin deaminases of E.coli (Invivogen) and S.cerevisiae (Genebank)
-
no consensus found at the amino acid-sequence  
+
no consensus found in the amino acid-sequence  
discussion at the next meeting
discussion at the next meeting
<br>
<br>
-
===3. Labday 15.04.2010===
+
===<p style="font-size:17px; background-color:#00dd77;">3. Labday 15.04.2010</p>===
====<p style="font-size:15px; background-color:#66bbFF;"><b>Theoretical cloning</b></p>====
====<p style="font-size:15px; background-color:#66bbFF;"><b>Theoretical cloning</b></p>====
Line 183: Line 77:
<ul>
<ul>
-
<li>pAAV-MCS vector (Stratagene): we survey the vector for igEM-restiction sites (RFC-25)<br>
+
<li>pAAV-MCS vector (Stratagene): we checked the vector for igEM-restiction sites: (RFC-25)<br>
'''EcoRI''' 1327; <b>Xbal</b> 1344; are inside the MCS <br>
'''EcoRI''' 1327; <b>Xbal</b> 1344; are inside the MCS <br>
<b>NgoMIV</b> 2254 are inside the V1 Ori region<br>
<b>NgoMIV</b> 2254 are inside the V1 Ori region<br>
<br>
<br>
-
<li>function of the Beta-Globin intron ( pAAV MCS Vector): increases the expression rate in Vivo/ Vitro with several mechanisms ( i.a. increased m-RNA accumulation <br>
+
<li>function of the Beta-Globin intron ( pAAV MCS Vector): increases the expression rate in Vivo/ Vitro through several mechanisms ( i.a. increased m-RNA accumulation <br>
-
([[Media: Freigem10_Nott_et_al_A_quantitative_analysis_of_intron_effects_on_mammalian_gene_expression_2003.pdf?]]; <br> [[Media: Freigem10_Haddad_et_al_A_systematic_study_of_the_function_of_the_h-beta-globin_introns_on_the_expression_2009.pdf?]])<br>
+
<br>
<br>
<li> Thymidin Kinase : we received another modified thymídin kinase-sequence from Amor, to do: check for iGEM restriction sites and develop a cloning strategie  
<li> Thymidin Kinase : we received another modified thymídin kinase-sequence from Amor, to do: check for iGEM restriction sites and develop a cloning strategie  
Line 195: Line 88:
<br>
<br>
-
===4. Labday 22.04.2010===
+
===<p style="font-size:17px; background-color:#00dd77;">4. Labday 22.04.2010</p>===
====<p style="font-size:15px; background-color:#66bbFF;"><b>Theoretical cloning</b></p>====
====<p style="font-size:15px; background-color:#66bbFF;"><b>Theoretical cloning</b></p>====
<p><b>Investigators: Adrian, Chris W., Hanna, Bea</b></p><br>
<p><b>Investigators: Adrian, Chris W., Hanna, Bea</b></p><br>
-
 
-
 
*tomorrow we will obtain the vector sequence for the thymidinkinase (WT) from Amor
*tomorrow we will obtain the vector sequence for the thymidinkinase (WT) from Amor
*call at Stratagene: #240071 AAV-Helper Free System, 1412 € (netto), available until next week ; we asked for a discount (Bea got Infos per E-mail)
*call at Stratagene: #240071 AAV-Helper Free System, 1412 € (netto), available until next week ; we asked for a discount (Bea got Infos per E-mail)
Line 207: Line 98:
<br>
<br>
-
===5. Labday 23.04.2010===
+
===<p style="font-size:17px; background-color:#00dd77;">5. Labday 23.04.2010</p>===
====<p style="font-size:15px; background-color:#66bbFF;"><b>Theoretical cloning</b></p>====
====<p style="font-size:15px; background-color:#66bbFF;"><b>Theoretical cloning</b></p>====
Line 214: Line 105:
<p><b>Investiators: Adrian, Chris W., Hanna, Kerstin, Anissa, (Kristian, Tobias, Sven)</b></p><br>
<p><b>Investiators: Adrian, Chris W., Hanna, Kerstin, Anissa, (Kristian, Tobias, Sven)</b></p><br>
-
*thymidin kinase from Amor: there is a PstI restriction site within the sequence (instead of this kinase we will probably utilize the TK30 + SR39 mutant -> we have to find their sequences )
+
*thymidin kinase from Amor: there is a PstI restriction site within the sequence (instead of this kinase we will probably use the TK30 + SR39 mutant -> we have to find their sequences )
-
*analyze of the secundary struckture of the ITR's (pAAV MCS of Stratagene), to decide if we can perform a point mutation to delete the PstI-restriction site :  
+
*analysis of the ITR secondary structure (pAAV MCS of Stratagene), to decide if we can introduce a point mutation to delete the PstI-restriction site :  
-
http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/8/82/ITRright.pdf <br>
+
[[Media:Freiburg10_ITRright.pdf| Right ITR]]<br>
-
http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/4/48/ITRleft_ohnePstI.pdf <br>
+
[[Media:Freiburg10_ITRleft.pdf| Left ITR]]<br>
http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/e/ef/Freiburg_10ITRleft.pdf <br>
http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/e/ef/Freiburg_10ITRleft.pdf <br>
http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/b/b5/Freiburg_10ITRright_AAV2.pdf <br>
http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/b/b5/Freiburg_10ITRright_AAV2.pdf <br>
*sequence alignement of the beta-globin of the pAAV MCS Plasmid with human beta-globin:  
*sequence alignement of the beta-globin of the pAAV MCS Plasmid with human beta-globin:  
-
http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/8/83/Freiburg10_Nucleotide_alignment_221_extraction.pdf <br>
+
[[Media:Freiburg10_Nucleotide alignment of the AAV.pdf| Nucleotide alignment of the AAV]]
-
*To do: gather informations about the mutant thymidinkinase (TK30 + SR39)  
+
<br>
-
 
+
*To do: gather information about the mutant thymidinkinase (TK30 + SR39)  
-
we won't use this kinase...
+
 +
<center>[https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May '''=> Go to Labjournal May (labday 6 - 17)''']</center><br>
<html>
<html>
</div>
</div>
</html>
</html>

Latest revision as of 21:39, 27 October 2010

=> Back to Notebook overview

NoteBook Navigator


Contents

2. Labday 13.04.2010

The Biobricks Foundation: RFC

Investigators: Chris W., Bea, Kira, Hanna, Julian, Anna, Jessica (Tobias,Sven, Kristian)


The BioBricks Foundation is dedicated to promoting and protecting open development, sharing, and reuse of BioBrick™ standard biological parts.
Homepage: http://openwetware.org/wiki/The_BioBricks_Foundation:RFC

Two important standards:

BBF RFC-10:
This standard defines the required sequence properties for a Biobrick(tm) standard biological part.
http://dspace.mit.edu/bitstream/handle/1721.1/45138/BBFRFC10.txt?sequence=1

BBF RFC-25: Fusion Protein (Freiburg) Biobrick assembly standard
This Request for Comments (RFC) describes an extension to the original BioBrick assembly standard (BBF RFC 10).
Media:Freiburg10 BBF RFC 25.pdf


Theoretical cloning
DNA- and protein-analyse:

  • Thymidin Kinase HSVI

Accesion: V00470 (Genebank) CDS without iGEM-restriction sites, unkown sequence in front of CDS with an EcoRI-restriction site, sequence from 1980! (There is a lot of X-ray crystallography analysis data available, but just to be on the safe side we should check the sequence again)

  • Cytosine Deaminase (see also FCY1) (S.cerevisae)

Accession number: AF005261 (Genebank) CDS without iGEM-restriction site, unkown in front of CDS with PstI-restriction site, sequence from 1997

  • pORF-CodA::upp (E.coli) [Vektor from InvivoGen]

CDS of the Cytosine Deaminase (E.coli) without iGEM-restriction site

  • Alignment of the amino acid-sequences of the cytosin deaminases of E.coli (Invivogen) and S.cerevisiae (Genebank)

no consensus found in the amino acid-sequence discussion at the next meeting

3. Labday 15.04.2010

Theoretical cloning

Investigators: Kira, Johannes, Bea


  • pAAV-MCS vector (Stratagene): we checked the vector for igEM-restiction sites: (RFC-25)
    EcoRI 1327; Xbal 1344; are inside the MCS
    NgoMIV 2254 are inside the V1 Ori region

  • function of the Beta-Globin intron ( pAAV MCS Vector): increases the expression rate in Vivo/ Vitro through several mechanisms ( i.a. increased m-RNA accumulation

  • Thymidin Kinase : we received another modified thymídin kinase-sequence from Amor, to do: check for iGEM restriction sites and develop a cloning strategie


4. Labday 22.04.2010

Theoretical cloning

Investigators: Adrian, Chris W., Hanna, Bea


  • tomorrow we will obtain the vector sequence for the thymidinkinase (WT) from Amor
  • call at Stratagene: #240071 AAV-Helper Free System, 1412 € (netto), available until next week ; we asked for a discount (Bea got Infos per E-mail)
  • next step (tomorrow, 10 am): modification of the multiple cloning site


5. Labday 23.04.2010

Theoretical cloning

Investiators: Adrian, Chris W., Hanna, Kerstin, Anissa, (Kristian, Tobias, Sven)


  • thymidin kinase from Amor: there is a PstI restriction site within the sequence (instead of this kinase we will probably use the TK30 + SR39 mutant -> we have to find their sequences )
  • analysis of the ITR secondary structure (pAAV MCS of Stratagene), to decide if we can introduce a point mutation to delete the PstI-restriction site :

Right ITR
Left ITR
http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/e/ef/Freiburg_10ITRleft.pdf
http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/b/b5/Freiburg_10ITRright_AAV2.pdf

  • sequence alignement of the beta-globin of the pAAV MCS Plasmid with human beta-globin:

Nucleotide alignment of the AAV
  • To do: gather information about the mutant thymidinkinase (TK30 + SR39)
=> Go to Labjournal May (labday 6 - 17)

Retrieved from "http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April"