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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 (BBa_K368001): Insertion [ ] -> Sequence of PCR1 from sequencing: confirmed [ ]
PCR3 (BBa_K368016): Insertion [X] -> Sequence of PCR3 from sequencing: confirmed [X]
PCR5 (BBa_K368017): Insertion [ ] -> Sequence of PCR5 from sequencing: confirmed [ ]
PCR6 (BBa_K368018): Insertion [ ] -> Sequence of PCR6 from sequencing: confirmed [ ]
PCR8 (BBa_K368019): 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
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