Team:Tuebingen/Notebook
Dronpa-Caged Cre
Ligation of gene blocks (Cre, Cre_sn, Dronpa, HCV, HCV+linker, nluc), linker L6, L9, L12, L15, nDegron and NLS with pSB1C3 and transformation in DH10beta:
Colony PCR and test restriction to check for correct transformation
pSB-Cre_sn and pSB-Luciferase did not yield correct colonies (transformation repeated), the other constructs resulted in correct colonies
Cre Reporter Cassette
Ligation of gene blocks (tADH, loxP WT, loxP66, loxP71) with pSB1C3 and transformation in DH10beta
pRS Vectors
Mutagenesis primer design pRS313, pRS315, pRS316
Dronpa-Caged Cre
Colony PCR of pSB-luc
Transformation of pSB-Gal and pSB-luc in DH10beta
Three part assembly of Linker(6/9/12/15) + Cre, Linker(6,9,12,15) + Dronpa with pTUM100 and transformation in yeast resulting in mixed colony PCR results (L6-Cre, L9-Cre, L12-Cre and L9-Dronpa yielded correct bands, the other assamblies did not result in correct fragment lengths)
Dronpa-Caged Cre
Three part assembly of NLS+Dronpa and Linker 6/9/12/15+Cre_sn with pTUM104
Transformation of L9-Cre, pTUM-L12-Dronpa, pTUM-L15_Dronpa and L15-Cre in yeast
Colony PCR of pTUM-L12_Cre (no successs), pTUM-L9-Cre, pTUM-L12-Dronpa (no successs), pTUM-L15_Dronpa (no successs), pTUM-L6-Dronpa, pTUM-L9-Dronpa (no successs)
Cre Reporter Cassette
Three part assembly of RFP+tADH,loxP+nluc and pADH+ loxP with pTUM100 (no positive results)
Dronpa-Caged Cre
Colony PCR of pTUM-L9-Dronpa, pTUM-L15-Dronpa (no successs), pTUM-L15_Cre
Three part assemblies: NLS+Dronpa, L12+Dronpa with pTUM104; L9+Cre, L15+Cre, L12+Cre with pTUM100
Two part assemblies: L6+Cre_sn, L9+Cre_sn, L12+Cre_sn, L15+Cre_sn, pTUM104+Dronpa, pTUM104-Cre, pTUM104+Cre_sn,
Transformation of all assemblies only successful with pTUM104-L12-Dronpa
Cre Reporter Cassette
Three part assembly: RFP+tADH, loxP+nluc and pADH+loxP with pTUM100
Two part assemblies: pTUM104+nluc, pTUM104+luc
Dronpa-Caged Cre
Control restriction and transformation of pTUM100 and pTUM104 for trouble shooting
Two part assemblies:L9-Cre, L15-Cre, L12-Dronpa with pTUM100; three part assembly: Dronpa+NLS with pTUM104
Colony PCR of pTUM100 and pTUM104 primers
pRS Vectors
Mutagenesis and transformation of pRS313 and pRS316 (both not successful)
Interlabstudy
Transformation of J23101 and J23106 in pSB1C3
Dronpa-Caged Cre
Ligation and transformation of assemblies: Cre, Cre_sn, Dronpa with pTUM 104, Dronpa with pSB1C3, L9-Cre-L9-Dronpa, L9-Cre_sn-L9-Dronpa, L12-Cre-L12-Dronpa, L12-Cre_sn-L12-Dronpa
Retransformation of pSB-Dronpa
Colony PCR of pTUM104-Cre, pTUM104-Cre_sn, pTUM104-Dronpa, pSB1C3-Cre_ns, pTUM100-L9-Cre_sn, pTUM100-L15-Cre_sn
Cre Reporter Cassette
Ligation and transformation of assemblies: RFP-tADH, nluc, luc (not successful) with pTUM104, pADH-loxP, loxP-nluc with pTUM100
Retransformation of pSB-loxP-WT and pSB-nluc
Colony PCR of pTUM104-RFP-tADH, pTUM104-loxP-nluc, pTUM104-pADH-loxP, pTUM104-nluc, pTUM104-luc
Interlabstudy
Colony PCR of pSB1C3-J23101, pSB1C3-J23106, pSB1C3-J23101+I13504, pBS1C3-J23106+I13504, pSB1C3-J23117+I13504
Transformation of J23151 and TetR repressible promotor (positive control)
Ligation and transformation of pSB1C3-J23101+I13504 (not successful), pBS1C3-J23106+I13504(not successful), pSB1C3-J23117+I13504
Dronpa-Caged Cre
Ligation and transformation of assemblies: pTUM100-L6-Dronpa, pTUM100-L15-Dronpa
Plate-reader fluorescence measurement: Dronpa and Sec61-GFP worked as intended, RFP and WT measurements seemed off
Cre Reporter Cassette
Ligation and transformation of pSB1C3-pADH-loxP-RFP-tADH (not successful)
pRS Vectors
Mutagenesis PCR of pRS313, pRS315, pRS316 (no success), overhang PCR of tADH
Interlabstudy
Ligation and transformation of pSB1C3-J23101+I13504, pBS1C3-J23106+I13504
Colony PCR of pSB1C3-J23101(not successful), pSB1C3-J23106, pSB1C3-J23117
Dronpa-Caged Cre
Fluorescence microscopy of yeast cells with transformed plamids: Sec61-GFP (positive control), pTUM104-Dronpa, pTUM104-NLS-Donpa, pTUM104-loxp-RFP & WT (negative control)
Ligation and transformation of pTUM104-L6-Cre-L6-Dronpa, pTUM104-L6-Cre_sn-L6-Dronpa, pTUM104-L12-Cre-L12-Dronpa, pTUM104-L12-Cre_sn-L12-Dronpa, pTUM104-L15-Cre-L15-Dronpa, pTUM104-L15-Cre_sn-L15-Dronpa
Colony PCR of pTUM104-L6-Cre-L6-Dronpa, pTUM104-L6-Cre_sn-L6-Dronpa, pTUM104-L12-Cre-L12-Dronpa, pTUM104-L12-Cre_sn-L12-Dronpa, pTUM104-L15-Cre-L15-Dronpa, pTUM104-L15-Cre_sn-L15-Dronpa
Sequencing of Cre_sn results in 8bp deletion (wrong synthesis by IDT), part is no longer used for further cloning
Cre Reporter Cassette
Ligation and transformation of pSB1C3-pADH-loxP-RFP-tADH (not successful)
pRS Vectors
Newly acquired pRS315 from local research group works properly
Mutagenesis PCR and transformation of pRS315 (successful) and pRS316 (not successful)
Ligation of pRS313 with MCS primer
Dronpa-Caged Cre
Transformation of pSB1C3-Cre
Ligation and transformation of Linker 6/9/12/15-Cre with pTUM100 (successful)
Cre Reporter Cassette
Transformation of pSB1C3-luc, pSB1C3-pADH-tADH
Ligation and transformation of pADH-loxP-RFP-tADH-loxP-nluc with pTUM100 (not successful), loxP-nluc with pTUM104 (successful), Dronpa with pETue(test expression in E. coli) (successful)
pRS Vectors
Colony PCR of pRS313-MCS (not successful), therefore new ligation and transformation of the construct
Sensors/Promotors
Transformation of pSB1C3-ENO2 (taken from iGEM registry)(successful)
Interlabstudy
New transformation of all interlab study constructs (positive control, negative control, promotors J23101-I13504, J23106-I13504, J23117-I13504 in pSB1C3)
Dronpa-Caged Cre
Colony PCR of pTUM100-L15-Cre
Ligation and transformation of L6/L9/L12/L15-Cre with L6/L9/L12/L15-Dronpa (successful)
Transformation of pSB1C3-Cre_sn (not sucessful) and ligation of L15-Cre-L15-Dronpa with pTUM104 (sucessful)
Ligation and transformation of pTUM104-NLS-Dronpa-L6-Cre-L6-Dronpa, pTUM104-NLS-Dronpa-L9-Cre-L9-Dronpa, pTUM104-NLS-Dronpa-L12-Cre-L12-Dronpa,
Colony PCR of pSB1C3-Cre_sn
Cre Reporter Cassette
Ligation of pTUM100-pADH-loxP-RFP-tADH-loxP-nluc (successful)
Ligation and transformation of alternative yeast vectors pYX122-mPR D.r. and pYX122-mPR X.I. with pADH-loxP-RFP-tADH-loxP-nluc
pRS Vectors
Mutagenisis PCR of pRS315 and pRS316
Transformation of mutated pRS315 and two times mutated pRS316 (both sucessful)
Sensors/Promotors
Ligation and transformation of pENO2-nluc, pENO2-RFP with pTUM100
Successful colony PCR of pTUM100-pENO2-nluc and pTUM100-pENO2-RFP
Ligation and transformation of pSUC/pFET (both taken form iGEM project Team Tuebingen 2013)/pADH+nluc/RFP with pTUM100
Colony PCR of pTUM100-pFET-nluc, pTUM100-pFET-RFP, pTUM100-pSUC-nluc, pTUM100-pSUC-RFP, pTUM100-pADH-nluc (all sucessful
2014 Part characterization
Transformation of pETue, pETue-intein-NAGA (successful) and pSB1K3-EABase (successful) in expression strain BL21(DE3) (all parts taken from Team Tuebingen's iGEM 2014 project)
Ligation of Dronpa with pETue (not sucessful)
Transformation of of pETue-Dronpa, pETue-Intein-EABase and pETue-Intein-NAGA in new competent BL21 (DE3) cells
Expression tests of Intein-NAGA and Intein-EABase yielding no expression
Colony PCR of pETue-Intein-NAGA and pETue-Dronpa
Dronpa-Caged Cre
Colony PCR of pTUM104-NLS-Dronpa-L15-Cre-L15-Dronpa (not successful)
Ligation and transformation of pSB1C3-NLS-Dronpa-L6-Cre-L6-Dronpa,pSB1C3-NLS-Dronpa-L9-Cre-L9-Dronpa, pSB1C3-NLS-Dronpa-L12-Cre-L12-Dronpa, pSB1C3-L6-Cre-L6-Dronpa
tADH Overhang PCR
Cre Reporter Cassette
Transformation of pYX122-pADH-loxP-RFP-tADH-loxP-nluc-pADH
Ligation and transformation of pSB1C3-pADH-loxP-RFP-tADH-loxP-nluc, pYX122-mPR D.r.
pRS Vectors
Ligation and transformation of pRS315-pADH-loxP-RFP-tADH-loxP-nluc-tADH, pRS316-NLS-Dronpa-L6-Cre-L6-Dronpa-tADH, pRS316-NLS-Dronpa-L9-Cre-L9-Dronpa-tADH, pRS316-NLS-Dronpa-L12-Cre-L12-Dronpa-tADH, pRS316-Dronpa-L6-Dronp-tADH, pRS315-MSC-pADH-loxP-RFP-tADH-loxP-nluc-pADH
Ligation of pRS315-MCS-pADH-loxP-RFP-tADH-loxP-nluc-tADH, pRS316-L6-Cre-L6-dronpa-tADH, pRS316-NLS-Dronpa-L6-Cre-L6-Dronpa-tADH, pRS316-NLS-Dronpa-L9-Cre-L9-Dronpa-tADH, pRS316-NLS-Dronpa-L12-Cre-L12-Dronpa-tADH
Sensors/Promotors
Microscopy and nanoluc-assay of pENO2-RFP, pFET-RFP, pSUC2-RFP (+galactose), pSUC2-RFP (+raffinose), pSU2-RFP (+glucose), pTUM100-pADH-loxP-RFP-tADH-loxP-nluc-pADH, pTUM100-pADH-nluc, pTUM100-pADH-loxP-RFP-tADH-loxP-nluc, pTUM100-SUC, pTUM104-nluc, WT-yeast
2014 Part characterization
Successful expression of Dronpa (in pETue) and Intein-NAGA (in pETue), therefore Ni-NTA purification and analysis via SDS page
Sensors/Promotors
Nanoluc-assay of pENO2-RFP, pFET-RFP, pSUC2-RFP (+galactose), pSUC2-RFP (+raffinose), pSU2-RFP (+glucose), pTUM100-pADH-loxP-RFP-tADH-loxP-nluc-pADH, pTUM100-pADH-nluc, pTUM100-pADH-loxP-RFP-tADH-loxP-nluc, pTUM100-SUC, pTUM104-nluc, WT-yeast