Difference between revisions of "Part:BBa K337038"

Revision as of 03:43, 27 October 2010

pSMB_miTuner Plasmid HD4 (BGH(rc)/shRNA10(rc)/RSV(rc)/CMV_TetO2/Luc2_sv40/CMV/Kozag_hRluc_BGH)

miTuner plasmid HD4 is used for microRNA binding site characterization and expression tuning.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1659
Illegal BamHI site found at 1
Illegal BamHI site found at 3734
Illegal XhoI site found at 3476
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1860
Illegal NgoMIV site found at 3204
Illegal NgoMIV site found at 3225
Illegal NgoMIV site found at 3497
Illegal AgeI site found at 2928
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 4913
Illegal SapI.rc site found at 3110

Characterization

Promoter efficiency of miRNA kit was checked in HEK T-Rex cells. Therefor 50ng of each construct with different promoter set-ups (table 1) were transfected into HEK 293 T-REx cells in 96-well plate format using FuGENE transfection reagent. As every construct is expressing firefly luciferase (luc2) and renilla luciferase (hRluc) at the same time the setup is unaffected by transfection efficiency and cell number variations. Each sample was transfected and measured by Dual luciferase assay in 8 replicates. As by this time no shRNA has been cloned into plasmid NO knock-down of luc2 is expected and the different expression efficiencies allow for characterization of the different promoters combinations. BBa_K337032 leads to a relative luciferase unit (RLU) of luc2 to hRluc expression of 6 RLU. BBa_K337035 and BBa_K337035 are showing a comparable expression of 12 - 13 RLU, which is in line with the knowledge that both luciferases are driven by the CMV promoter. Hek 293 T-Rex cells stably express the Tet repressor thus allows us to observe very efficient repression of Firefly luciferse expression if a CMV-TetO2 promoter is driving luc2 (BBa_K337038 and BBa_K337046). BBa_K337040 transfection into Hek T-Rex cells results in an expression of 15 RLU. BBa_K337042 and BBa_K337044 are constructed in a way that luc2 is driven by the CMV promoter and hRluc is driven by the RSV promoter and show a comparable expression of 17-20 RLU. This leads to the conclusion that the CMV promoter shows comparable expression to the RSV promoter in Hek T-Rex cell lines.

Table 1

part	promoter driving luc2 (Firefly)	promoter driving (Renilla)	promoter driving shRNA expression
BBa_K337032	RSV	CMV	SV40
BBa_K337035	CMV	CMV	SV40
BBa_K337036	CMV	CMV	RSV
BBa_K337038	CMV TetO2	CMV	RSV
BBa_K337040	RSV	RSV	SV40
BBa_K337042	CMV	RSV	SV40
BBa_K337044	CMV	RSV	RSV
BBa_K337046	CMV TetO2	RSV	RSV

Promoter strength characterization of tuning constructs in HEK 293 T-REx cell line

@@ Line 16: / Line 16: @@
 <partinfo>BBa_K337038 parameters</partinfo>
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+===Characterization===
 Promoter efficiency of miRNA kit was checked in HEK T-Rex cells. Therefor 50ng of each construct with different promoter set-ups (table 1) were transfected into HEK 293 T-REx cells in 96-well plate format using FuGENE transfection reagent. As every construct is expressing firefly luciferase (luc2) and renilla luciferase (hRluc) at the same time the setup is unaffected by transfection efficiency and cell number variations. Each sample was transfected and measured by Dual luciferase assay in 8 replicates. As by this time no shRNA has been cloned into plasmid NO knock-down of luc2 is expected and the different expression efficiencies allow for characterization of the different promoters combinations. [https://parts.igem.org/wiki/index.php?title=Part:BBa_K337032 BBa_K337032] leads to a relative luciferase unit (RLU) of luc2 to hRluc expression of 6 RLU. [https://parts.igem.org/wiki/index.php?title=Part:BBa_K337035 BBa_K337035] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K337035 BBa_K337035] are showing a comparable expression of 12 - 13 RLU, which is in line with the knowledge that both luciferases are driven by the CMV promoter.  Hek 293 T-Rex cells stably express the Tet repressor thus allows us to observe very efficient repression of Firefly luciferse expression if a CMV-TetO2 promoter is driving luc2 ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K337038 BBa_K337038] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K337046 BBa_K337046]). [https://parts.igem.org/wiki/index.php?title=Part:BBa_K337040 BBa_K337040] transfection into Hek T-Rex cells results in an expression of 15 RLU. [https://parts.igem.org/wiki/index.php?title=Part:BBa_K337042 BBa_K337042] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K337044 BBa_K337044] are constructed in a way that luc2 is driven by the CMV promoter and hRluc is driven by the RSV promoter and show a comparable expression of 17-20 RLU. This leads to the conclusion that the CMV promoter shows comparable expression to the RSV promoter in Hek T-Rex cell lines.
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