Team:Freiburg Bioware/testpage

From 2010.igem.org

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===2. Labday 13.04.2010===
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===1. Labday 31.03.2010===
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====<p style="font-size:15px; background-color:#66bbFF;"><b>The Biobricks Foundation: RFC</b></p>====
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====<p style="font-size:15px; background-color:#66bbff;"><b> Sequence analysis</b></p>====
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<p><b>Investigators: Chris W., Bea, Kira, Hanna, Julian, Anna, Jessica (Tobias,Sven, Kristian)</b></p><br>
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<b>Investigators: Christian W., Bea, Kira, Achim, Kerstin, Hanna, Christian L. (Tobias, Kristian)</b><br>
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The BioBricks Foundation is dedicated to promoting and protecting the open development, <br>
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'''Theoretical cloning'''
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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]]
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Two important standarts:<br>
 
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'''BBF RFC-10''':<br>
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Sequence analysis (Alignement) of the Stratagene Vector with
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This standard defines the required sequence properties for a Biobrick(tm) standard biological part.<br>
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*AAV2
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[[http://dspace.mit.edu/bitstream/handle/1721.1/45138/BBFRFC10.txt?sequence=1]]<br>
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*AAV5
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*AAV7
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*AAV8
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'''BBF RFC-25: Fusion Protein (Freiburg) Biobrick assembly standard'''<br>
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'''result:'''  
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This Request for Comments (RFC) describes an extension to the original BioBrick assembly standard (BBF RFC 10). <br>
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[[Media:AAV_alignment.pdf]]
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[[Media:Freiburg10 BBF RFC 25.pdf]]
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'''Theoretical cloning'''<br>
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DNA- and protein-analyse:
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*Thymidin Kinase HSVI <br>
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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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*Cytosine Deaminase (see also FCY1) ('''S.cerevisae''')<br>
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Accession number: AF005261 (Genebank)
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CDS without iGEM-restriction site, unkown in front of CDS with PstI-restriction site, sequence from 1997<br>
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*pORF-CodA::upp ('''E.coli''') [Vektor from InvivoGen]
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CDS of the Cytosine Deaminase (E.coli) without iGEM-restriction site
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*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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discussion at the next meeting
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<br>
<br>
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Curiously there were some significant differences in the aligned sequences of several AAV genomes
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resulting in frame shifts.
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Maybe because the sequences are from 1983 and therefore incomplete.
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In case of doubt we count on the Stratagene-sequence.
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===3. Labday 15.04.2010===
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'''To do:'''
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*download and compare of further AAV serotypes. (In Geneious)
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====<p style="font-size:15px; background-color:#66bbFF;"><b>Theoretical cloning</b></p>====
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*compare literature
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*find out which possibilities are given to modify the Capsid
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<p><b>Investigators: Kira, Johannes, Bea</b></p>
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<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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'''EcoRI''' 1327; <b>Xbal</b> 1344; are inside the MCS <br>
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<b>NgoMIV</b> 2254 are inside the V1 Ori region<br>
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<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 ([[Media: Freigem10_Nott_et_al_A_quantitative_analysis_of_intron_effects_on_mammalian_gene_expression_2003.pdf?]]; [[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>
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<li> Thymidin Kinase : we received another modified thymídin kinase-sequence from Amor, to do: check for iGEM restriction sites and develop a cloning strategie
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</ul>
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<br>
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===4. Labday 22.04.2010===
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====<p style="font-size:15px; background-color:#66bbFF;"><b>Theoretical cloning</b></p>====
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<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
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*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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*next step (tomorrow, 10 am): modification of the  multiple cloning site
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<br>
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===5. Labday 23.04.2010===
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====<p style="font-size:15px; background-color:#66bbFF;"><b>Theoretical cloning</b></p>====
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<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 )
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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 : http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/8/82/ITRright.pdf http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/images/4/48/ITRleft_ohnePstI.pdf 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
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*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
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*To do: gather informations about the mutant thymidinkinase (TK30 + SR39)
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we won't use this kinase...
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Revision as of 10:46, 6 September 2010

1. Labday 31.03.2010

Sequence analysis

Investigators: Christian W., Bea, Kira, Achim, Kerstin, Hanna, Christian L. (Tobias, Kristian)


Theoretical cloning


Sequence analysis (Alignement) of the Stratagene Vector with

  • AAV2
  • AAV5
  • AAV7
  • AAV8

result: Media:AAV_alignment.pdf
Curiously there were some significant differences in the aligned sequences of several AAV genomes resulting in frame shifts. Maybe because the sequences are from 1983 and therefore incomplete. In case of doubt we count on the Stratagene-sequence.

To do:

  • download and compare of further AAV serotypes. (In Geneious)
  • compare literature
  • find out which possibilities are given to modify the Capsid

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