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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.
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
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