Team:Freiburg Bioware/NoteBook/Labjournal/April

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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
  (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?)
  
  
• 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)