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

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Latest revision as of 14:32, 25 October 2010


	Cut'N'Survive System Functional Principle Sequences Schedule Jump-or-Die System Functional Principle Sequences Schedule ProSearch System Schedule

	Functional Principle Sequences Schedule picture gallery

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

@@ Line 1: / Line 1: @@
 {{:Team:LMU-Munich/Templates/Page Header}}
+[[Image:cnsZelle.png|300px|Logo|right]]
-==Aim 1: DNA reproduction+PCR==
-==Aim 2: Assembling Biobricks==
-==Aim 3: Testing products==
-==Aim 4: Testing system==
-oder
-[[Team:LMU-Munich/Cut'N'survive/Schedule/Aim 1|Aim 1: DNA reproduction+PCR]]
+==Aim 1: DNA replication+PCR==
+For PCR key see [[Team:LMU-Munich/Cut'N'survive/Schedule/PCR key | PCR key]]
-[[Team:LMU-Munich/Cut'N'survive/Schedule/Aim 2|Aim 2: Assembling Biobricks]]
+<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>
-[[Team:LMU-Munich/Cut'N'survive/Schedule/Aim 3|Aim 3: Testing products]]
-[[Team:LMU-Munich/Cut'N'survive/Schedule/Aim 4|Aim 4: Testing system]]
+<b>PCR1: replication of the Tet-inducible CMV promotor   [X]</b>
+[[Image:PCR1.jpg]]
+Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer1| 1TreF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer2| 2TreR]]
+expected product size: 492bp
-alt:
+<b>PCR2a: replication of the TEVrecogn-NDegron-SF3 Part with mutagenisis (EcoR1 + Pst1) [X]</b>
+[[Image:PCR2a.jpg]]
-Meilensteine
+Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer3| 3TevF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer4| 4TevMutEPR]]
+expected product size: 332 bp
-Für die Projekte haben wir uns jeweils folgende „Meilensteine“ gesetzt:
-<p><b>„TEV-System“</b></p>
-. 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€
+<b>PCR2b: replication of the TEVrecogn-NDegron-SF3 Part with mutagenisis (EcoR1 + Pst1) [X]</b>
+[[Image:PCR2b.jpg]]
-. Verknüpfen von eGFP als Zielgen mit einer TEV-Schnittstelle, einer TEV-Protease, und interagierendem Protein B. (Nachweis: eGFP leuchtet, Proteinchromatographie)
+Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer5| 5TevMutEPF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer6| 6TevR]]
+expected product size: 772 bp
-Aufwand: ca. 6-8 Wochen, 2000€, Parallele Arbeit ist möglich und notwendig
-. Einbringen beider Konstrukte in Zellen (Nachweis: eGFP leuchtet, einige Zellen sterben, Proteinchromatographie)
+<b>PCR3: joining PCR of the TEVrecogn-NDegron-SF3 Part [X]</b>
+[[Image:PCR3.jpg]]
-Aufwand: ca. 2 Wochen, 1000€
+Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer3| 3TevF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer6| 6TevR]]
+expected product size: 1087 bp
+<b>PCR4a: replication human bak with mutagenisis (Pst1) [X]</b>
+[[Image:PCR4a.jpg]]
+Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer7| 7BakF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer8| 8BakMutPR]]
+expected product size: 330 bp
+<b>PCR4b: replication human bak with mutagenisis (Pst1) [X]</b>
+[[Image:PCR4b.jpg]]
+Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer9| 9BakMutPF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer10| 10BakR]]
+expected product size: 376 bp
+<b>PCR5: joining PCR of human bak [X]</b>
+[[Image:PCR5.jpg]]
+Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer7| 7BakF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer10| 10BakR]]
+expected product size: 688 bp
+<b>PCR6: replication of the SV40-polyadenylation site [X]</b>
+[[Image:PCR6.jpg]]
+Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer11| 11PAF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer12| 12PAR]]
+expected product size: 237 bp
+<b>PCR7a: replication of the p14*TEVrecogn with mutagenisis (Spe1) [X]</b>
+[[Image:PCR7a.jpg]]
+Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer13| 13TrecF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer14| 14TrecMutSR]]
+expected product size: 850 bp
+<b>PCR7b: replication of the p14*TEVrecogn with mutagenisis (Spe1) with TEV recogn in primer (16TrecR) [X]</b>
+[[Image:PCR7b.jpg]]
+Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer15| 15TrecMutSF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer16| 16TrecR]]
+expected product size: 402 bp
+<b>PCR8: joining PCR of p14*TEVrecogn with TEV recogn in primer (16TrecR) [X]</b>
+[[Image:PCR8.jpg]]
+Primer used:[[Team:LMU-Munich/Cut'N'survive/Schedule/Primer13| 13TrecF]][[Team:LMU-Munich/Cut'N'survive/Schedule/Primer16| 16TrecR]]
+expected product size: 1233 bp
+==Aim 2: Inserting PCR Products in pSB1C3 and verifying Sequence ==
+PCR1 (BBa_K368001): Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR1| Sequence of PCR1 from sequencing]]: confirmed [ ]
+PCR3 (BBa_K368016): Insertion [X] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR3| Sequence of PCR3 from sequencing]]: confirmed [X]
+PCR5 (BBa_K368017): Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR5| Sequence of PCR5 from sequencing]]: confirmed [ ]
+PCR6 (BBa_K368018): Insertion [ ] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR6| Sequence of PCR6 from sequencing]]: confirmed [ ]
+PCR8 (BBa_K368019): Insertion [X] -> [[Team:LMU-Munich/Jump-or-die/Schedule/SequenzPCR8| Sequence of PCR8 from sequencing]]: confirmed [X]
+==Aim 3: Assembling Biobricks==
+We are using the [[Team:LMU-Munich/Notebook/Protocols/13_3A_Method_for_Biobrick_assembly|3A System]] to assemble Biobricks.
+Assembled Biobricks:
+* BB7 (BBa_K368007): assembled [ ]; tet-on promoter (prev TRE CMV) + TEV recognition site + SF3b155;
+[[Team:LMU-Munich/Jump-or-die/Schedule/SequenzBB7| Sequence of BB7 from sequencing]] confirmed: [ ]
+* BB8 (BBa_K368008): assembled [ ]; Human Bak + SV40PA ;
+[[Team:LMU-Munich/Jump-or-die/Schedule/SequenzBB8| Sequence of BB8 from sequencing]]  confirmed: [ ]
+* BB9 [BBa_K368009): assembled [ ]; tet-on promoter (prev TRE CMV) + TEV recognition site + SF3b155 + Human Bak + SV40PA;
+[[Team:LMU-Munich/Jump-or-die/Schedule/SequenzBB9| Sequence of BB9 from sequencing]]  confirmed: [ ]
+* BB10 (BBa_K368010): assembled [ ]; CMV-promoter + TEV protesase + p14* + TEV recognition site ;
+[[Team:LMU-Munich/Jump-or-die/Schedule/SequenzBB10| Sequence of BB10 from sequencing]]  confirmed: [ ]
+* BB11 (BBa_K368011): assembled [X]; eGFP + SV40PA ;
+[[Team:LMU-Munich/Jump-or-die/Schedule/SequenzBB11| Sequence of BB11 from sequencing]]  confirmed: [X]
+* BB12 (BBa_K368012): assembled [ ]; CMV-promoter + TEV protesase + p14* + TEV recognition site + eGFP + SV40PA;
+[[Team:LMU-Munich/Jump-or-die/Schedule/SequenzBB12| Sequence of BB12 from sequencing]]  confirmed: [ ]
+<b> Assembling Construct 1 </b>
+[[Image: N1S.jpg]]
+<b> Assembling Construct 2 </b>
+[[Image: N2S.jpg]]
+==Aim 4: Testing products==
+=== Construct 1 ===
+[[Image:Konstruktcut1.png|400pxs|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 ===
+[[Image:Konstruktcut2.png|400pxs|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==
-. Parallel wird versucht, das erste Konstrukt stabil ins Zellgenom einzubauen.
-Aufwand: ca. 2 Wochen, 500€
 {{:Team:LMU-Munich/Templates/Page Footer}}