04/06 - 10/06

  • Golden-Gate-Cloning-Reaction (GGC) to assemble a 12-repeat-TAL-TF, that recognizes the sequence TACATTGGACCTAT
  • transformation: GGC-reaction
  • making aliquots of ordered GGC-Primers (freiGEM-method)
  • making aliquots of ordered, i.e. synthesized, direpeats
  • extension-PCR of direpeats in order to produce diefferent restriction sites for correct location of direpeats
  • PCR-Purification of extension-PCR
  • mutagenesis-PCR of Zhang-plasmid to remove restriction sites (SpeI, PstI, EcoRI, XbaI) --> making the plasmid work with iGEM-standard

11/06 - 16/06

  • optimizing PCR conditions for extension of direpeats
  • redoing GGC-reaction
  • transformation of redone GGC-reaction
  • GGC á la freiGEM --> transformation
  • testing of iGEM-distribution kit

17/06 - 23/06

  • cloning of direpeats into pJET 1.2 vector-system
  • colony-PCR of freiGEM-GGC product
  • place sequencing order for freiGEM-GGC

24/06 - 01/07

  • optimizing of freiGEM-GGC under various conditions
  • transformation of pJET-direpeats into bacteria
  • using the iGEM distribution kit: Bba_A12564 (CMV+Luc) --> reporter-plasmid for DNA-methyl-transferase testing
  • Miniprep of GGC-transformation (1 successful)

02/07 - 08/07

  • extension-PCR with all 96 direpeats on one well-plate
  • transformation
  • PCR-amplification of all 4 iGEM-backbones --> testing different conditions
  • making bacteria competent for transformation

09/07 - 15/07

  • digest of exDirepeats with XbaI and PstI
  • nanodrop of exDirepeats
  • ligation of exDirepeats in psB1C3 vector backbone and then transformation
  • colony-PCR, gel run, making cultures
  • miniprep of some of the 96 exDirepeats

16/07 - 22/07

  • transformation of synthesis products (Dnmt, hyperactive Gin, Gin L7C7, Tn3 and TAL-ORF) into DH10B-strain competent cells
  • amplification of psb1c3 vector backbone, then gel-run and gel-purification
  • picking of colonies (transformation of synthesis-products)
  • miniprep of synthesis-products
  • repeat of pcr-amplification of psb1c3 vector backbone

30/07 - 05/08

  • GGC to produce freiGEM-Mammo-Brick with CMV-promotor, Puromycin-resistance and postORF and TALEN-NG-vector backbone
  • to do so a extension-PCR on the parts was done
  • CMV-Promotor was taken out of iGEM Distribution Kit 2012

06/08 - 12/08

  • mutagenesis-PCR of pcb1c3 vector backbone to eliminate restriction site for BsmBI
  • repeat of GGC to get MammoBrick
  • gel-run of mutagenesis-PCR of psb1c3

13/08 - 19/08

  • repeating the assembly of MammoBrick vector containing CMV Promotor, PostORF-Part and Puromycin-ORF
  • repeating mutagenesis-PCR on vector backbone psb1c3
  • extPCR of TAL-ORF (synthesis-product) in order to produce a n-terminal kind of adapter to paste recombinase
  • producing an provisory mammalian expression vector --> cloning of TAL-ORF into mammalian vector backbone
  • to do so the vector and the insert have been digested and then ligated and transformed into dh10b e. coli strain bacteria
  • transformation of mutated vector backbone
  • colony pcr to test provisory mammalian expression vector

20/08 - 26/08

  • repeating mutagenesis pcr for vector backbone psb1c3 with another template
  • finding a new method for successful extension pcr and then digest and ligation into the psb1cr backbone

27/08 - 02/09

  • finding out that CMV promotor taken out of the registry distribution kit is not good at all
  • trying to get another vector with cmv promotor and ordering new primer pair to amplify it
  • after finding a working method for direpeat extension, digestion and ligation into psb1c3 vector backbone we now started to execute it on our synthesized direpeats

03/09 - 09/09

  • GGC-reaction in order to produce recombinase-linker-n-tal-orf construct
  • annealing of linker-part (was ordered as two single-strand primers)
  • after ggc-reaction was done a pcr-amplification on the new assembled part was executed
  • gel-run shows a very weak band at 2,7 kb for hyperactive gin recombinase
  • GGC-reaction in order to clone direpeats for a complete TAL-Domain into provisory mammalian expression vector
  • repeating the extextPCR on the recombinase-linker-n-tal-orf construct under different conditions after the first pcr reaction failed
  • amplification of mutated psb1c3 vector backbone
  • trying to clone Transcription Factor into provisory mammalian expression vector
  • repeating of GGC reaction in order to produce MammoBrick
  • still working on our toolkit, doing the final steps: sent them off for sequencing

10/09 - 16/09

  • doing the final steps in producing our toolkit: miniprep was done for the last three batches of direpeats, which can finally be sequenced on monday
  • we now have 75 successfully sequenced direpeats in the psb1c3 vector backbone, ready to send them off to igem headquarters
  • new working cmv promotor is now in psb1c3 vector backbone, too. we now have an improved, i.e. finally working, biobrick for the registry

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