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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) [X]
Primer used: 7BakF 8BakMutPR
expected product size: 330 bp
PCR4b: replication human bak with mutagenesis (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
PCR9: replication of eGFP with attP in primer [X]
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
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