Team:LMU-Munich/Cut'N'survive/Schedule

Aim 1: DNA replication+PCR

For PCR key see PCR key

Colourcode for the primers: Annealing part, mutagenised part, other sequences integrated into primer

PCR1: replication of the Tet-inducible CMV promotor [X]

Primer used: 1TreF 2TreR expected product size: 492bp

PCR2a: replication of the TEVrecogn-NDegron-SF3 Part with mutagenisis (EcoR1 + Pst1) [X]

Primer used: 3TevF 4TevMutEPR expected product size: 332 bp

PCR2b: replication of the TEVrecogn-NDegron-SF3 Part with mutagenisis (EcoR1 + Pst1) [X]

Primer used: 5TevMutEPF 6TevR expected product size: 772 bp

PCR3: joining PCR of the TEVrecogn-NDegron-SF3 Part [X]

Primer used: 3TevF 6TevR expected product size: 1087 bp

PCR4a: replication human bak with mutagenisis (Pst1) [X]

Primer used: 7BakF 8BakMutPR expected product size: 330 bp

PCR4b: replication human bak with mutagenisis (Pst1) [X]

Primer used: 9BakMutPF 10BakR expected product size: 376 bp

PCR5: joining PCR of human bak [X]

Primer used: 7BakF 10BakR expected product size: 688 bp

PCR6: replication of the SV40-polyadenylation site [X]

Primer used: 11PAF 12PAR expected product size: 237 bp

PCR7a: replication of the p14*TEVrecogn with mutagenisis (Spe1) [X]

Primer used: 13TrecF 14TrecMutSR expected product size: 850 bp

PCR7b: replication of the p14*TEVrecogn with mutagenisis (Spe1) with TEV recogn in primer (16TrecR) [X]

Primer used: 15TrecMutSF 16TrecR expected product size: 402 bp

PCR8: joining PCR of p14*TEVrecogn with TEV recogn in primer (16TrecR) [X]

Primer used: 13TrecF 16TrecR expected product size: 1233 bp

Aim 2: Inserting PCR Products in pSB1C3 and verifying Sequence

PCR1: Insertion [ ] -> Sequence of PCR1 from sequencing: confirmed [ ]

PCR3: Insertion [X] -> Sequence of PCR3 from sequencing: confirmed [X]

PCR5: Insertion [ ] -> Sequence of PCR5 from sequencing: confirmed [ ]

PCR6: Insertion [ ] -> Sequence of PCR6 from sequencing: confirmed [ ]

PCR8: Insertion [X] -> Sequence of PCR8 from sequencing: confirmed [X]

Aim 3: Assembling Biobricks

We are using the 3A System to assemble Biobricks.

Assembled Biobricks:

• BB7 (BBa_K368007): assembled [ ]; tet-on promoter (prev TRE CMV) + TEV recognition site + SF3b155;

Sequence of BB7 from sequencing confirmed: [ ]

• BB8 (BBa_K368008): assembled [ ]; Human Bak + SV40PA ;

Sequence of BB8 from sequencing confirmed: [ ]

• BB9 [BBa_K368009): assembled [ ]; tet-on promoter (prev TRE CMV) + TEV recognition site + SF3b155 + Human Bak + SV40PA;

Sequence of BB9 from sequencing confirmed: [ ]

• BB10 (BBa_K368010): assembled [ ]; CMV-promoter + TEV protesase + p14* + TEV recognition site ;

Sequence of BB10 from sequencing confirmed: [ ]

• BB11 (BBa_K368011): assembled [X]; eGFP + SV40PA ;

Sequence of BB11 from sequencing confirmed: [X]

• BB12 (BBa_K368012): assembled [ ]; CMV-promoter + TEV protesase + p14* + TEV recognition site + eGFP + SV40PA;

Sequence of BB12 from sequencing confirmed: [ ]

Assembling Construct 1

Assembling Construct 2

Aim 4: Testing products

Construct 1

- transform into HeLa cells to see if they survive.

-> Check the leakiness of tet-on-promoter

- induce tet-on-promoter to see, if cells die.

-> Check if construct 1 is working

Construct 2

- Transform into HeLa cells and see if eGFP is being translated

-> Check if CMV-promoter is working and construct is being read-off completely

- Western Blot

-> Check by reference of protein size if eGFP is cut off from the residual of the protein by TEV-Protease

Aim 5: Testing system