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From 2012.igem.org

Notebook

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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 --> protocol: sanjana et al.

• 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