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

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[[image:Jumplogo.png|250px|right|ApoControl logo]]
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==Aim 1: DNA reproduction+PCR==
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For PCR key see [[Team:LMU-Munich/Cut'N'survive/Schedule/PCR key | PCR key]]
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<b>Colourcode for the primers: <font color="#FF0000">Annealing part</font>, <font color="#009933">mutagenised part</font>, <font color="#FF6600">other sequences integrated into primer</font></b>
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<b>PCR1: replication of the Tet-inducible CMV promotor  [X]</b>
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[[Image:PCR1.jpg]]
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Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer1| 1TreF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer2| 2TreR]]
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expected product size: 492 bp
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<b>PCR4a: replication human bak with mutagenesis (Pst1) [X]</b>
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[[Image:PCR4a.jpg]]
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Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer7| 7BakF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer8| 8BakMutPR]]
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expected product size: 330 bp
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<b>PCR4b: replication human bak with mutagenesis (Pst1) [X]</b>
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[[Image:PCR4b.jpg]]
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Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer9| 9BakMutPF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer10| 10BakR]]
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expected product size: 376 bp
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<b>PCR5: joining PCR of human bak [X]</b>
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[[Image:PCR5.jpg]]
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Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer7| 7BakF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer10| 10BakR]]
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expected product size: 688 bp
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<b>PCR6: replication of the SV40-polyadenylation site [X]</b>
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[[Image:PCR6.jpg]]
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Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer11| 11PAF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer12| 12PAR]]
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expected product size: 237 bp
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<b>PCR9: replication of eGFP with attP in primer [X]</b>
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[[Image:PCR9.jpg]]
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Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer20| 20eGFPattPF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer21| 21eGFPR]]
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expected product size: 808 bp
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<b>PCR10: Replikation of PhiC31o [X]</b>
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[[Image:PCR10.jpg]]
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Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer22| 22PC31oF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer23| 23PC31oR]]
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expected product size: 1888 bp
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<b>Note: for attB two Primers ([[Team:LMU-Munich/Cut'N'survive/Schedule/Primer18| 18attBF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer19| 19attBR]]) used.</b>
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==Aim 2: Inserting PCR Products in pSB1C3 and verifying Sequence ==
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PCR1 (BBa_K368001): Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR1| Sequence of PCR1 from sequencing]]: confirmed [ ]
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PCR5 (BBa_K368017): Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR5| Sequence of PCR5 from sequencing]]: confirmed [ ]
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PCR6 (BBa_K368018): Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR6| Sequence of PCR6 from sequencing]]: confirmed [ ]
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PCR9 (BBa_K368021): Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR9| Sequence of PCR9 from sequencing]]: confirmed [ ]
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PCR10 (BBa_K368022): Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR10| Sequence of PCR10 from sequencing]]: confirmed [ ]
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==Aim 3: Assembling Biobricks==
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We are using the [[Team:LMU-Munich/Notebook/Protocols/13_3A_Method_for_Biobrick_assembly|3A System]] to assemble Biobricks.
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Assembly of BioBricks:
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* BB1 ( BBa_K368024): Assembly [ ]; prevTRE CMV + attB site
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-> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenceBB1| Sequence of BB1 from sequencing]]: confirmed [ ]
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* BB2 ( BBa_K368002): Assembly [ ]; Human Bak + SV40PA
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-> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenceBB2| Sequence of BB2 from sequencing]]: confirmed [ ]
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* BB3 ( BBa_K368003): Assembly [ ]; prevTRE CMV+ attB site + Human Bak + SV40PA
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-> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenceBB3| Sequence of BB3 from sequencing]]: confirmed [ ]
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* BB4 ( BBa_K368004): Assembly [X]; eGFP + SV40PA
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-> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenceBB4| Sequence of BB4 from sequencing]]: confirmed [X]
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* BB5 ( BBa_K368005): Assembly [ ]; PhiC31o + SV40PA
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-> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenceBB5| Sequence of BB5 from sequencing]]: confirmed [ ]
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* BB6 ( BBa_K368006): Assembly [ ]; CMV-promoter + PhiC31o + SV40PA
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-> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenceBB6| Sequence of BB6 from sequencing]]: confirmed [ ]
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<b> Assembling Construct 1</b>
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[[Image: J1S.jpg]]
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<b> Assembling Construct 2</b>
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[[Image: J2S.jpg]]
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<b> Assembling Construct 3</b>
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[[Image: J3S.jpg]]
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==Aim 4: Testing products==
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=== Construct 1 ===
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[[Image: Konstruktjump1.PNG | 400pxs | Jump construct 1]]
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- Transform into HeLa cells to see if they survive.
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-> Check the leakiness of tet-on-promoter
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- Induce tet-on-promoter to see, if cells die.
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-> Check if construct 1 is working
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=== Construct 2 ===
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[[Image: Konstruktjump2.PNG | 400pxs | Jump construct 2]]
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- Sequencing
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=== Construct 3 ===
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[[Image: Konstruktjump3.PNG | 400pxs | Jump construct 3]]
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- Transform into HeLa cells
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- Western Blot or Proteinchromatography
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-> Check if PhiC31o is read off
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==Aim 5: Testing system==
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[[image:Jumplogo.png|200px|center|ApoControl logo]]
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{{:Team:LMU-Munich/Templates/Page Footer}}
{{:Team:LMU-Munich/Templates/Page Footer}}

Latest revision as of 14:44, 25 October 2010


ApoControl logo


Contents

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] PCR1.jpg

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


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

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


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

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


PCR5: joining PCR of human bak [X] PCR5.jpg

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


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

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


PCR9: replication of eGFP with attP in primer [X] PCR9.jpg

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


PCR10: Replikation of PhiC31o [X] PCR10.jpg

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

J1S.jpg

Assembling Construct 2

J2S.jpg

Assembling Construct 3

J3S.jpg

Aim 4: Testing products

Construct 1

Jump 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

Jump construct 2

- Sequencing

Construct 3

Jump construct 3

- Transform into HeLa cells

- Western Blot or Proteinchromatography

-> Check if PhiC31o is read off

Aim 5: Testing system

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