iGEM 2015 — Team Tuebingen

# Team:Tuebingen/Notebook

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## 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