Team:LMU-Munich/Cut'N'survive/Schedule

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(New page: {{:Team:LMU-Munich/Templates/Page Header}} Meilensteine Für die Projekte haben wir uns jeweils folgende „Meilensteine“ gesetzt: <p><b>„TEV-System“</b></p> 1. Verknüpfen von Bak...)
 
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{{:Team:LMU-Munich/Templates/Page Header}}
{{:Team:LMU-Munich/Templates/Page Header}}
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Meilensteine
 
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Für die Projekte haben wir uns jeweils folgende „Meilensteine“ gesetzt:
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[[Image:cnsZelle.png|300px|Logo|right]]
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<p><b>„TEV-System“</b></p>
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1. Verknüpfen von Bak mit interagierendem Protein A, einem N-Degron und einer Schnittstelle für die TEV-Protesase, sodass Bak noch aktiv bleibt. Davor ist ein induzierbarer Promotor und eine Antibiotikaresistenz geschaltet (Nachweis: induzierbarer Zelltod) Aufwand: ca. 6-8 Wochen, 2000€
 
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2. Verknüpfen von eGFP als Zielgen mit einer TEV-Schnittstelle, einer TEV-Protease, und interagierendem Protein B. (Nachweis: eGFP leuchtet, Proteinchromatographie)
 
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Aufwand: ca. 6-8 Wochen, 2000€, Parallele Arbeit ist möglich und notwendig
 
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3. Einbringen beider Konstrukte in Zellen (Nachweis: eGFP leuchtet, einige Zellen sterben, Proteinchromatographie)
 
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Aufwand: ca. 2 Wochen, 1000€
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==Aim 1: DNA replication+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: 492bp
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 +
 
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<b>PCR2a: replication of the TEVrecogn-NDegron-SF3 Part with mutagenisis (EcoR1 + Pst1) [X]</b>
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[[Image:PCR2a.jpg]]
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Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer3| 3TevF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer4| 4TevMutEPR]]
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expected product size: 332 bp
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 +
 
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<b>PCR2b: replication of the TEVrecogn-NDegron-SF3 Part with mutagenisis (EcoR1 + Pst1) [X]</b>
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[[Image:PCR2b.jpg]]
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Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer5| 5TevMutEPF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer6| 6TevR]]
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expected product size: 772 bp
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<b>PCR3: joining PCR of the TEVrecogn-NDegron-SF3 Part [X]</b>
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[[Image:PCR3.jpg]]
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Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer3| 3TevF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer6| 6TevR]]
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expected product size: 1087 bp
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 +
 
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<b>PCR4a: replication human bak with mutagenisis (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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 +
 
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<b>PCR4b: replication human bak with mutagenisis (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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 +
 
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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>PCR7a: replication of the p14*TEVrecogn with mutagenisis (Spe1) [X]</b>
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[[Image:PCR7a.jpg]]
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Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer13| 13TrecF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer14| 14TrecMutSR]]
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expected product size: 850 bp
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<b>PCR7b: replication of the p14*TEVrecogn with mutagenisis (Spe1) with TEV recogn in primer (16TrecR) [X]</b>
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[[Image:PCR7b.jpg]]
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Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer15| 15TrecMutSF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer16| 16TrecR]]
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expected product size: 402 bp
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<b>PCR8: joining PCR of p14*TEVrecogn with TEV recogn in primer (16TrecR) [X]</b>
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[[Image:PCR8.jpg]]
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Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer13| 13TrecF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer16| 16TrecR]]
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expected product size: 1233 bp
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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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PCR3 (BBa_K368016): Insertion [X] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR3| Sequence of PCR3 from sequencing]]: confirmed [X]
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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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PCR8 (BBa_K368019): Insertion [X] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR8| Sequence of PCR8 from sequencing]]: confirmed [X]
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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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Assembled Biobricks:
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* BB7 (BBa_K368007): assembled [ ]; tet-on promoter (prev TRE CMV) + TEV recognition site + SF3b155;
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[[Team:LMU-Munich/Jump-or-die/Schedule/SequenzBB7| Sequence of BB7 from sequencing]] confirmed: [ ]
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* BB8 (BBa_K368008): assembled [ ]; Human Bak + SV40PA ;
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[[Team:LMU-Munich/Jump-or-die/Schedule/SequenzBB8| Sequence of BB8 from sequencing]]  confirmed: [ ]
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* BB9 [BBa_K368009): assembled [ ]; tet-on promoter (prev TRE CMV) + TEV recognition site + SF3b155 + Human Bak + SV40PA;
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[[Team:LMU-Munich/Jump-or-die/Schedule/SequenzBB9| Sequence of BB9 from sequencing]]  confirmed: [ ]
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* BB10 (BBa_K368010): assembled [ ]; CMV-promoter + TEV protesase + p14* + TEV recognition site ;
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[[Team:LMU-Munich/Jump-or-die/Schedule/SequenzBB10| Sequence of BB10 from sequencing]]  confirmed: [ ]
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* BB11 (BBa_K368011): assembled [X]; eGFP + SV40PA ;
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[[Team:LMU-Munich/Jump-or-die/Schedule/SequenzBB11| Sequence of BB11 from sequencing]]  confirmed: [X]
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* BB12 (BBa_K368012): assembled [ ]; CMV-promoter + TEV protesase + p14* + TEV recognition site + eGFP + SV40PA;
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[[Team:LMU-Munich/Jump-or-die/Schedule/SequenzBB12| Sequence of BB12 from sequencing]]  confirmed: [ ]
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<b> Assembling Construct 1 </b>
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[[Image: N1S.jpg]]
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<b> Assembling Construct 2 </b>
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[[Image: N2S.jpg]]
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==Aim 4: Testing products==
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=== Construct 1 ===
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[[Image:Konstruktcut1.png|400pxs|TEV Construct 1: Inserted in celline]]
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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:Konstruktcut2.png|400pxs|TEV Construct 2: Contains gene of interest]]
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- Transform into HeLa cells and see if eGFP is being translated
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-> Check if CMV-promoter is working and construct is being read-off completely
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- Western Blot
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-> Check by reference of protein size if eGFP is cut off from the residual of the protein by TEV-Protease
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==Aim 5: Testing system==
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4. Parallel wird versucht, das erste Konstrukt stabil ins Zellgenom einzubauen.
 
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Aufwand: ca. 2 Wochen, 500€
 
{{:Team:LMU-Munich/Templates/Page Footer}}
{{:Team:LMU-Munich/Templates/Page Footer}}

Latest revision as of 14:32, 25 October 2010


Logo



Contents

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] PCR1.jpg

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


PCR2a: replication of the TEVrecogn-NDegron-SF3 Part with mutagenisis (EcoR1 + Pst1) [X] PCR2a.jpg

Primer used: 3TevF 4TevMutEPR expected product size: 332 bp


PCR2b: replication of the TEVrecogn-NDegron-SF3 Part with mutagenisis (EcoR1 + Pst1) [X] PCR2b.jpg

Primer used: 5TevMutEPF 6TevR expected product size: 772 bp


PCR3: joining PCR of the TEVrecogn-NDegron-SF3 Part [X] PCR3.jpg

Primer used: 3TevF 6TevR expected product size: 1087 bp


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

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


PCR4b: replication human bak with mutagenisis (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


PCR7a: replication of the p14*TEVrecogn with mutagenisis (Spe1) [X] PCR7a.jpg

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] PCR7b.jpg

Primer used: 15TrecMutSF 16TrecR expected product size: 402 bp


PCR8: joining PCR of p14*TEVrecogn with TEV recogn in primer (16TrecR) [X] PCR8.jpg

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

N1S.jpg

Assembling Construct 2

N2S.jpg

Aim 4: Testing products

Construct 1

TEV Construct 1: Inserted in celline

- 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

TEV Construct 2: Contains gene of interest

- 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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