Team:Heidelberg/Notebook natural promoters
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=='''Natural Promoters'''== | =='''Natural Promoters'''== | ||
=='''Contents'''== | =='''Contents'''== | ||
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== 9-26-2009 == | == 9-26-2009 == | ||
* Miniprep of the HSP70 promoter/p31 construct. | * Miniprep of the HSP70 promoter/p31 construct. | ||
- | * Test digest (NheI and EcoRI) of RARE, c-Jun, LDLR and HMG CoA construct at 37°C for one hour. | + | * Test digest (NheI and EcoRI) of RARE, c-Jun, LDLR and HMG CoA construct at 37°C for one hour (Fig. 6-10). |
* Following constructs were send for sequencing: | * Following constructs were send for sequencing: | ||
C-Jun #19, c-Jun #20, c-Jun #21, c-Jun #22, RARE #8, RARE #9, RARE #15, RARE #23, HMG CoA #24, HMG CoA #26, LDLR #3, LDLR #5, LDLR #11, LDLR #12, LDLR #19, LDLR #20, LDLR #21, LDLR #22, LDLR #24, LDLR #25, LDLR #26 and HMG CoA #35 . | C-Jun #19, c-Jun #20, c-Jun #21, c-Jun #22, RARE #8, RARE #9, RARE #15, RARE #23, HMG CoA #24, HMG CoA #26, LDLR #3, LDLR #5, LDLR #11, LDLR #12, LDLR #19, LDLR #20, LDLR #21, LDLR #22, LDLR #24, LDLR #25, LDLR #26 and HMG CoA #35 . | ||
- | [[image:HD09_Jun2.png|center|600px|thumb|'''Figure | + | [[image:HD09_Jun2.png|center|600px|thumb|'''Figure 6: 1% agarose gel electrophoresis of a test digest of the c-Jun/p31 plasmid constructs.'''The c-Jun/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (100 - |
10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting c-Jun insert should be 341 bp long. C-Jun #19, c-Jun #20, c-Jun #21 and c-Jun #22 seem to be successfully ligated plasmids, because there are small bands at ~ 300 bp. All lanes show a p31 plasmid band, which is 4919 bp long. But other c-Jun inserts could not be recognized and so all other lanes display definetly the wrong product (JeT, ~180 bp).]] | 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting c-Jun insert should be 341 bp long. C-Jun #19, c-Jun #20, c-Jun #21 and c-Jun #22 seem to be successfully ligated plasmids, because there are small bands at ~ 300 bp. All lanes show a p31 plasmid band, which is 4919 bp long. But other c-Jun inserts could not be recognized and so all other lanes display definetly the wrong product (JeT, ~180 bp).]] | ||
- | [[image:HD09_RARE.png|center|600px|thumb|'''Figure | + | [[image:HD09_RARE.png|center|600px|thumb|'''Figure 7: 1% agarose gel electrophoresis of a test digest of the RARE/p31 plasmid constructs.'''The RARE/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (100 - |
10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting RARE insert should be 212 bp long. The RARE inserts are difficult to recognize, because the JeT insert of the original plasmid is ~ 180 bp. Therefore, some good constructs are chosen: RARE #8, RARE #9, RARE #15 and RARE #23.]] | 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting RARE insert should be 212 bp long. The RARE inserts are difficult to recognize, because the JeT insert of the original plasmid is ~ 180 bp. Therefore, some good constructs are chosen: RARE #8, RARE #9, RARE #15 and RARE #23.]] | ||
- | [[image:HD09_HMGCoA.png|center|600px|thumb|'''Figure | + | [[image:HD09_HMGCoA.png|center|600px|thumb|'''Figure 8: 1% agarose gel electrophoresis of a test digest of the HMG CoA synthase/p31 plasmid constructs.'''The HMG CoA synthase/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (100 - |
10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting HMG CoA synthase insert should be 363 bp long. All lanes show a p31 plasmid band, which is 4919 bp long. But the HMG CoA inserts could not be recognized. All lanes display definetly the wrong product (JeT, ~180 bp).]] | 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting HMG CoA synthase insert should be 363 bp long. All lanes show a p31 plasmid band, which is 4919 bp long. But the HMG CoA inserts could not be recognized. All lanes display definetly the wrong product (JeT, ~180 bp).]] | ||
- | [[image:HD09_LDL_HMG.png|center|600px|thumb|'''Figure | + | [[image:HD09_LDL_HMG.png|center|600px|thumb|'''Figure 9: 1% agarose gel electrophoresis of a test digest of the LDLR/p31 and HMG CoA synthase/p31 plasmid constructs.'''The LDLR/p31 and HMG CoA synthase/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (100 - |
10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting LDLR insert should be 363 bp long and HMG CoA 363 bp. HMG CoA #24, HMG CoA #26, LDLR #3, LDLR #5, LDLR #11 and LDLR #12 seem to be successfully ligated plasmids, because there are small bands at ~ 350 bp. All lanes show a p31 plasmid band, which is 4919 bp long. But other HMG CoA and LDLR inserts could not be recognized. All other lanes display definetly the wrong product (JeT, ~180 bp).]] | 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting LDLR insert should be 363 bp long and HMG CoA 363 bp. HMG CoA #24, HMG CoA #26, LDLR #3, LDLR #5, LDLR #11 and LDLR #12 seem to be successfully ligated plasmids, because there are small bands at ~ 350 bp. All lanes show a p31 plasmid band, which is 4919 bp long. But other HMG CoA and LDLR inserts could not be recognized. All other lanes display definetly the wrong product (JeT, ~180 bp).]] | ||
- | [[image:HD09_HMG_LDL_2.png|center|600px|thumb|'''Figure | + | [[image:HD09_HMG_LDL_2.png|center|600px|thumb|'''Figure 10: 1% agarose gel electrophoresis of a test digest of the LDLR/p31 and HMG CoA synthase/p31 plasmid constructs.'''The NFkB/p31 and NFAT/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (100 - |
10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting LDLR insert should be 363 bp long and HMG CoA 363 bp. LDLR #19, LDLR #20, LDLR #21, LDLR #22, LDLR #24, LDLR #25, LDLR #26 and HMG CoA #35 seem to be successfully ligated plasmids, because there are small bands at ~ 350 bp. All lanes show a p31 plasmid band, which is 4919 bp long. But other HMG CoA and LDLR inserts could not be recognized. All other lanes display definetly the wrong product (JeT, ~180 bp).]] | 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting LDLR insert should be 363 bp long and HMG CoA 363 bp. LDLR #19, LDLR #20, LDLR #21, LDLR #22, LDLR #24, LDLR #25, LDLR #26 and HMG CoA #35 seem to be successfully ligated plasmids, because there are small bands at ~ 350 bp. All lanes show a p31 plasmid band, which is 4919 bp long. But other HMG CoA and LDLR inserts could not be recognized. All other lanes display definetly the wrong product (JeT, ~180 bp).]] | ||
== 9-28-2009 == | == 9-28-2009 == | ||
* Right constructs of RARE, c-Jun, LDLR and HMG CoA are: c-Jun #19, c-Jun #20, c-Jun #21, c-Jun #22, RARE #8, RARE #23, LDLR #19, LDLR #20, LDLR #21 and HMG CoA #35. | * Right constructs of RARE, c-Jun, LDLR and HMG CoA are: c-Jun #19, c-Jun #20, c-Jun #21, c-Jun #22, RARE #8, RARE #23, LDLR #19, LDLR #20, LDLR #21 and HMG CoA #35. | ||
- | * Test digest (NheI and EcoRI) of HSP70 at 37°C for one hour. | + | * Test digest (NheI and EcoRI) of HSP70 at 37°C for one hour (Fig. 11). |
* Following construct is send for sequencing: HSP70 #9. | * Following construct is send for sequencing: HSP70 #9. | ||
- | [[image:HD09_HSP70.png|center|600px|thumb|'''Figure | + | [[image:HD09_HSP70.png|center|600px|thumb|'''Figure 11: 1% agarose gel electrophoresis for a test digest of the HSP70/p31 plasmid construct.'''The HSP70/p31 plasmids were digested with EcoRI and PstI at 37°C for one hour. The numbers (1,3,4,5 etc.) describe the plasmids of different bacterial colonies. Additionally, a DNA ladder (100 - |
10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting HSP70 insert should be 402 bp long. HSP70 #9 seem to be a successfully ligated plasmid, because there is a small bands at ~ 400 bp. All lanes show a p31 plasmid band, which is 4919 bp long. But other HSP70 inserts could not be recognized and so all other lanes display definetly the wrong product (JeT, ~180 bp).]] | 10000 bp; Fermentas DNA Ladder Mix) is added into the gel for the validation of the DNA product length. The resulting HSP70 insert should be 402 bp long. HSP70 #9 seem to be a successfully ligated plasmid, because there is a small bands at ~ 400 bp. All lanes show a p31 plasmid band, which is 4919 bp long. But other HSP70 inserts could not be recognized and so all other lanes display definetly the wrong product (JeT, ~180 bp).]] | ||
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* Right constructs of HSP70 is: HSP70 #9. | * Right constructs of HSP70 is: HSP70 #9. | ||
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== 10-12-2009 == | == 10-12-2009 == | ||
- | * TECAN measurement of the natural LDL receptor promoter and the HMG CoA synthase promoter. | + | * TECAN (plate reader) measurement of the natural LDL receptor promoter and the HMG CoA synthase promoter. These promoters, which are coupled to GFP, were cotransfected with a reference plasmid including JeT coupled to mCherry and were induced by Lipoprotein Deficient Serum (See: [[Team:Heidelberg/Notebook_promoters_cells| Cell Culture]]). |
+ | [[image:HD09_LDL_HMG_CoA.png|center|400px|thumb|'''Figure 12: The HMG CoA synthase promoter and the LDL receptor promoter induced approximately 25-35% by Lipoprotein Deficient Serum.''' The LDL receptor promoter and HMG CoA synthase promoter is coupled to the fluorecent protein GFP. This GFP fluorescence was measured by TECAN once (à three replicates). Background fluorescence was substracted, and fluorescence levels are plotted relative to JeT. The standard deviations are indicated by the error bars.]] | ||
+ | '''Notebook information about the measurement of natural promoters is found in the Notebook [[Team:Heidelberg/Notebook_measure/NotebookFC| Measurement]] and [[Team:Heidelberg/Notebook_promoters_cells| Cell Culture]].''' | ||
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Latest revision as of 11:43, 20 October 2009
Natural PromotersContents
8-11-2009
8-18-2009
8-19-2009
8-20-2009
8-21-2009
8-24-2009
8-25-2009
8-26-2009
8-27-2009
8-28-2009
8-29-2009
8-31-2009
9-01-2009
9-02-2009
9-03-2009
9-04-2009
9-07-2009
9-08-2009
9-09-2009
9-10-09
9-11-09
9-14-2009
9-15-2009
9-16-2009
9-17-2009
9-18-2009
9-20-2009
9-21-2009
9-22-2009
9-23-2009
9-24-2009
9-25-2009
9-26-2009
C-Jun #19, c-Jun #20, c-Jun #21, c-Jun #22, RARE #8, RARE #9, RARE #15, RARE #23, HMG CoA #24, HMG CoA #26, LDLR #3, LDLR #5, LDLR #11, LDLR #12, LDLR #19, LDLR #20, LDLR #21, LDLR #22, LDLR #24, LDLR #25, LDLR #26 and HMG CoA #35 . 9-28-2009
9-29-2009
10-12-2009
Notebook information about the measurement of natural promoters is found in the Notebook Measurement and Cell Culture. |