Team:LMU-Munich/Jump-or-die/Schedule

Aim 1: DNA reproduction+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: 492 bp

PCR4a: replication human bak with mutagenesis (Pst1) [ ]

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

PCR4b: replication human bak with mutagenesis (Pst1) [ ]

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

PCR5: joining PCR of human bak [ ]

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

PCR9: replication of eGFP with attP in primer [ ]

Primer used: 20eGFPattPF 21eGFPR expected product size: 808 bp

PCR10: Replikation of PhiC31o [X]

Primer used: 22PC31oF 23PC31oR expected product size: 1888 bp

Note: for attB two Primers ( 18attBF 19attBR) used.

Aim 2: Inserting PCR Products in pSB1C3 and verifying Sequence

PCR1 (BBa_K368001): Insertion [ ] -> Sequence of PCR1 from sequencing: confirmed [ ]

PCR5 (BBa_K368017): Insertion [ ] -> Sequence of PCR5 from sequencing: confirmed [ ]

PCR6 (BBa_K368018): Insertion [ ] -> Sequence of PCR6 from sequencing: confirmed [ ]

PCR9 (BBa_K368021): Insertion [ ] -> Sequence of PCR9 from sequencing: confirmed [ ]

PCR10 (BBa_K368022): Insertion [ ] -> Sequence of PCR10 from sequencing: confirmed [ ]

Aim 3: Assembling Biobricks

We are using the 3A System to assemble Biobricks.

Assembly of BioBricks:

• BB1 ( BBa_K368024): Assembly [ ]; prevTRE CMV + attB site

-> Sequence of BB1 from sequencing: confirmed [ ]

• BB2 ( BBa_K368002): Assembly [ ]; Human Bak + SV40PA

-> Sequence of BB2 from sequencing: confirmed [ ]

• BB3 ( BBa_K368003): Assembly [ ]; prevTRE CMV+ attB site + Human Bak + SV40PA

-> Sequence of BB3 from sequencing: confirmed [ ]

• BB4 ( BBa_K368004): Assembly [X]; eGFP + SV40PA

-> Sequence of BB4 from sequencing: confirmed [X]

• BB5 ( BBa_K368005): Assembly [ ]; PhiC31o + SV40PA

-> Sequence of BB5 from sequencing: confirmed [ ]

• BB6 ( BBa_K368006): Assembly [ ]; CMV-promoter + PhiC31o + SV40PA

-> Sequence of BB6 from sequencing: confirmed [ ]

Assembling Construct 1

Assembling Construct 2

Assembling Construct 3

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

- Sequencing

Construct 3

- Transform into HeLa cells

- Western Blot or Proteinchromatography

-> Check if PhiC31o is read off

Aim 5: Testing system