Team:Freiburg Bioware/NoteBook/Labjournal/April

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<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March">March (labday 1)</a></li>
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<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April">April (labday 2 - 5)</a></li>
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<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May">May (labday 6 - 17)</a></li>
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<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August">August part 1 (labday 76 - 92)</a></li>
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<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August2">August part 2 (labday 93 - 106)</a></li>
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<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li>
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<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 149 )</a></li>
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<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 150 - 166 )</a></li>
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<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November  (labday 167 - 170 )</a></li>
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<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture</a></li>
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===<p style="font-size:17px; background-color:#00dd77;">2. Labday 13.04.2010</p>===
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===2. Labday 13.04.2010===
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====<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>
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The BioBricks Foundation is dedicated to promoting and protecting open development, sharing, and reuse of BioBrick™ standard biological parts.<br>
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The BioBricks Foundation is dedicated to promoting and protecting the open development, <br>
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sharing, and reuse of BioBrick™ standard biological parts.<br>
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Homepage: [[http://openwetware.org/wiki/The_BioBricks_Foundation:RFC]]
Homepage: [[http://openwetware.org/wiki/The_BioBricks_Foundation:RFC]]
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Two important standarts:<br>
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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)
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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)
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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)
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no consensus found at the amino acid-sequence  
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no consensus found in the amino acid-sequence  
discussion at the next meeting
discussion at the next meeting
<br>
<br>
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===3. Labday 15.04.2010===
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===<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>
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<li>pAAV-MCS vector (Stratagene): we survey the vector for igEM-restiction sites (RFC-25)<br>
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<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>
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<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>
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<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>
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([[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>
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<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>
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===4. Labday 22.04.2010===
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===<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>
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*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)
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<br>
<br>
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===5. Labday 23.04.2010===
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===<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>
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*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 )
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*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>
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[[Media:Freiburg10_ITRright.pdf| Right ITR]]<br>
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http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/4/48/ITRleft_ohnePstI.pdf <br>
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[[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:  
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http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/8/83/Freiburg10_Nucleotide_alignment_221_extraction.pdf <br>
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[[Media:Freiburg10_Nucleotide alignment of the AAV.pdf| Nucleotide alignment of the AAV]]
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*To do: gather informations about the mutant thymidinkinase (TK30 + SR39)  
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<br>
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*To do: gather information about the mutant thymidinkinase (TK30 + SR39)  
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we won't use this kinase...
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<center>[https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May '''=> Go to Labjournal May (labday 6 - 17)''']</center><br>
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Latest revision as of 21:39, 27 October 2010

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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"