Team:Heidelberg/Notebook natural promoters

Natural Promoters

Contents

plasmid p31 = plasmid pSMB_MEASURE

8-11-2009

• Plasmids and primers with the specicfic natural promoters had been ordered

8-18-2009

• Start with natural promoter project
• The two promoters, Apolipoprotein A-IV (APOAIV) and Cytochrome P450 1A1 (CYP1A1) promoter, were amplified by PCR using human genomic DNA.

8-19-2009

• APOAIV promoter PCR product was digested with NheI and SpeI for one hour.
• The backbone plasmid (p31; ~5000 bp) was also digested with the same restriction enzymes (1 hour) and it was subsequent digested with SAP (1 hour).

8-20-2009

• CYP1A1 promoter has the internal restriction site, NheI,...waiting for another CYP1A1 reverse primer with the restriction site PstI instead of NheI.
• The plasmid (p31) and the insert (APOAIV promotor) were purified and ligated at 22 °C for 3 hours.
• The ligated product was transformed into DH5alpha cells.

8-21-2009

• Picked eight colonies of p31/APOAIV plates and did a mini-preparation (miniprep)
• Test digest of the eight miniprep's using the restriction enzymes SpeI and NheI

IMAGE of digest

• There is an appropriate band fpr the APOAIV promoter (~4000bp length).
• Miniprep of colony p31/ApoAIV #1, p31/ApoAIV #2 and p31/ApoAIV #3

8-24-2009

• The plasmids p31/APOAIV #3 and p31/APOAIV #1 will be sequenced by GATC

8-25-2009

• APOAIV sequence did not match the published sequence
• The test digest did not yield expected fragments--> we need to troubleshoot and repeat PCR from genomic DNA

8-26-2009

• Troubleshooting PCR for CYP1A1 and APOAIV using Taq mastermix and Phu with freshly mixed ingredients. We also used various MgCl2 concentrations (1, 2, 3, 4 ul of 25 mM MgCl2 per 50ul reaction) and performed the reaction with and without the addition of DMSO.
• Analytic gel below shows CYP1A1 with Taq -/+ DMSO in the first two lanes with bands at the expected height of around 1200 bp. Lanes 1-10 are different CYP1A1 reactions, lanes 11-20 are APOAIV reactions. APOAIV shows only nonspecific bands, has to be further optimized using gradient PCR or new primers.

8-27-2009

• Test digest of CYP1A1 insert (1.2 kb) with NheI (digest at position 614 bp)
• PCR of NFkB responsive and NFAT responsive promoter plasmids to amplify promoters from Oakes plasmids p46 and p47. Amplified using PCR high annealing protocol:

8-28-2009

• Digest of CYP1A1 insert with PstI and SpeI and NFkB and NFAT inserts with PstI and EcoRI
• Digest of plasmid p31 with PstI/SpeI and PstI/EcoRI with SAP digest afterward
• PCR purification of digested inserts, gel purification of plasmid digests

8-29-2009

• Ligation of p31 and promoters (NFAT, NFkB)
• Transformation of ligated plasmids (p31) with the promoters NFAT and NFkB.

8-31-2009

• Miniprep of p31 plasmid + NFAT/NFkB promoter
• Ligation of CYP1A1 promoter and p31 plasmid
• Test digest of NFAT/NFkB promoter within the p31 plasmid (figure below)
• NFAT and NFkB promoters sent to sequencing

Figure ??: 1% agarose gel electrophoresis for a test digest of the NFkB/p31 plasmid and the NFAT/p31 plasmid. 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) is added into the gel for the validation of the DNA product length. The resulting NFAT insert should be 134 bp long and NFkB 179 bp. NFAT #2 and NFAT #5 seem to be successfully ligated plasmids, because there are small bands at ~ 150 bp. The lanes of NFkB #4 and NFkB #5 show a p31 plasmid band, which is 4919 bp long. But the NFkB inserts could not be recognized. Nevertheless, we will try to sequence them. All other lanes display definetly the wrong products.

9-01-2009

• Transformation of CYP1A1/p31 plasmid

9-02-2009

• Minipreparation of CYP1A1 colonies (six colonies)
• Test digest of the ligated CYP1A1 construct -> no success
• APOAIV promoter: PCR of genomic DNA with old conditions

9-03-2009

• Analysis with agarose gelelectrophoresis of the APOAIV-PCR product -> no success
• Digest of CYP1A1 promoter and plasmid p31 with the restriction enzymes, SpeI and PstI
• PCR Purification of the CYP1A1 promoter and gel extraction of p31.
• Ligation of digested CYP1A1 promoter and p31 (over night) at 16°C.

9-04-2009

• PCR with the plasmid p52 to isolate the HSP70 promoter
• PCR with the plasmid p46 to isolate the promoter with NFkB binding sites
• Digest of the plasmid p31 with the restriction enzymes, EcoRI and NheI

9-07-2009

• Digest of promoter with NFkB binding sites (NFkB_prom) with NheI and EcoRI
• Digest of half of the volume of the HSP70 promoter with EcoRI and NheI
• Digest of half of the volume of the HSP70 promoter with PstI and SpeI
• Ligation of the digested NFkB_prom and the HSP70 promoter (EcoRI, NheI) with p31 (EcoRI, NheI; 09/04/09)
• Ligation of the HSP70 promoter (EcoRI, NheI) with p31 (EcoRI, NheI; 09/04/09)
• Ligation of the HSP70 promoter (SpeI, PstI) with p31 (SpeI, PstI; 09/03/09)
• Transformation of the ligated products (above) and the ligated product of CYP1A1 (09/03/09)

9-08-2009

• Pick some colonies of transformed bacteria
• Miniprep
• Test digest

9-09-2009

• Gel electrophoresis image of the test digest:

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• CYP1A1_promoter/p31 construct was included in three positive colonies. Therefore this construct is ready for mutagenesis PCR to remove the NheI restriction site at 614 bp
• The others have not turned out satisfactory.

10-09-09

• The sequence of CYP1A1 #2 and #5 are correct.
• Plate out the addgene plasmids: pJC6-GL3, SBE4-Luc, pGL3-RARE-Luc, pGL3-NFAT-Luc, PUMA-Frag1-Luc, pLDLR-Luc, pHMGCS-Luc

11-09-09

• Pick some colonies of the plates (10.09.09) and miniprep.

14-09-2009

• PCR of HSP70, NFAT and NFkB
• Digest of the PCR products by NheI and EcoRI
• Digest of p31_BBB by NheI and EcoRI
• Send to sequencing: pJC6-GL3, SBE4-Luc, pGL3-RARE-Luc, pGL3-NFAT-Luc, PUMA-Frag1-Luc, pLDLR-Luc, pHMGCS-Luc
• Mutagenesis PCR of CYP1A1/p31 #5

15-09-2009

• PCR-Purification with HSP70, NFkB and NFAT
• Gel-extraction of p31_BBB, which was digested by NheI and EcoRI
• Ligation: Hsp70 with p31_BBB, NFAT with p31_BBB and NFkB with p31_BBB
• PCR troubleshooting with NFAT and NFkB
• Amplification of the p52 plasmid
• CYP1A1-muta/p31 PCR product was digested by DpnI
• Transformation of the three ligations (below: Hsp70/p31, NFAT/p31 and NFkB/p31) and CYP1A1-muta/p31

16-09-2009

• Arrival of the sequencing results of the ordered Addgene plasmids
• Design of primers for c-Jun promoter, LDL receptor promoter, Retinoic Acid Receptor Response Element
• Unfortunaltely, PUMA promoter is not used because of a PstI restrcition site and it is no time for mutagenesis PCR
• p52 (with HSP70 insert) is amplified in bacteria
• Miniprep of the following ligation : Hsp70 with p31_BBB, NFAT with p31_BBB and NFkB with p31_BBB
• And test digest of the ligations with EcoRI and PstI and there is something on the agarose gel
• The PCR troubleshooting with NFAT and NFkB were analysed by a agarose gel and there are not the right bands

BILD???

17-09-2009

• Miniprep of p52 (with HSP70 insert) and send to sequencing
• Ligation of NFAT and NFkB (of troubleshooting PCR) with p31 plasmid
• No colonies of CYP1A1-muta -> try again the mutagenesis PCR (with special mutagenesis Kit)

18-09-2009

• Design of primers for the HSP70 insert by analysis of p52 sequence
• Second mutagenesis PCR of CYP1A1
• Transformation of NFAT and NFkB ligations

20-09-2009

• pick colonies of NFAT and NFkB

21-09-2009

• PCR of RARE, LDLR, HMG CoA synthase, c-Jun
• Miniprep of NFAT and NFkB and test digest

22-09-2009

• Tranformation of CYP1A1-muta
• Analysis of the the following PCR products by agarose gel electrophoresis: RARE, LDLR, HMG CoA synthase, c-Jun.

BILD!!!

• Diegst of these PCR products by the restriction enzymes, NheI and EcoRI.

23-09-2009

• Gel extraction of RARE, LDLR, HMG CoA synthase and c-Jun insert.
• Ligation of these extracted inserts with the digested (NheI and EcoRI) plasmid p31.
• PCR of the p52 (with HSP70 promoter insert) and analysis of the PCR product by agarose gel electrophoresis.
• Digest of the HSP70 promoter by NheI and EcoRI.
• Transformation of the RARE/p31, LDLR/p31, c-Jun/p31 and HMG CoA synthase promoter/p31 construct.

24-09-2009

• Ligation of the HSP70 promoter with the digested (NheI and EcoRI) p31 plasmid.
• Transformation of the HSP70 promoter/p31 construct.
• Colonies of the RARE/p31, LDLR/p31, c-Jun/p31 and HMG CoA synthase promoter/p31 construct are picked.

25-09-2009

• Miniprep of RARE/p31, LDLR/p31, c-Jun/p31 and HMG CoA synthase promoter/p31 construct.
• Colonies of the HSP70 promoter/p31 construct are picked.

26-09-2009

• Miniprep of the HSP70 promoter/p31 construct.
• Test digest (NheI and EcoRI) of RARE, c-Jun, LDLR and HMG CoA synthase

28-09-2009

• Test diegst (NheI and EcoRI) of HSP70