Team:Heidelberg/Notebook natural promoters


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Contents

 *  plasmid p31 = plasmid pSMB_MEASURE 

8-11-2009

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

8-18-2009

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

8-19-2009

 * APOAIV promoter PCR product was digested with NheI and SpeI at 37°C for one hour.
 * The backbone plasmid (p31; ~5000 bp) was also digested with the same restriction enzymes (at 37°C, 1 hour) and it was subsequent digested with SAP (at 37°C, 1 hour). The insert of this plasmid p31 is the promoter JeT, which will be cut out after this digest.

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 (at 37°C, 1 hour)
 * There is an appropriate band for 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.

8-25-2009

 * APOAIV sequence did not match the published sequence (NCBI database). --> We have to 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 (Fig. 1). 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) at 37°C for one hour.
 * PCR of NFkB responsive and NFAT responsive promoter plasmids to amplify promoters from Oakes plasmids p46 and p47. Amplified using PCR high annealing protocol and primers with BBB_standard:

8-28-2009

 * Digest of CYP1A1 insert with PstI and SpeI and NFkB and NFAT inserts with PstI and EcoRI at 37°C for one hour.
 * Digest of plasmid p31 with PstI/SpeI and PstI/EcoRI with SAP digest afterward at 37°C for one hour each.
 * PCR purification of digested inserts, gel purification of plasmid digests.

8-29-2009

 * Ligation of p31 and promoters (NFAT, NFkB) at 22°C for 3 hours.
 * Transformation of ligated plasmids (p31) with the promoters NFAT and NFkB in DH5alpha cells.

8-31-2009

 * Miniprep of p31 plasmid + NFAT/NFkB promoter.
 * Ligation of CYP1A1 promoter and p31 plasmid at 22°C for 3 hours.
 * Test digest (EcoRI and PstI) of NFAT/NFkB promoter within the p31 plasmid (Fig. 2)at 37°C for one hour.
 * NFAT#2 and NFAT#5 plasmids sent to sequencing.

9-01-2009

 * Transformation of CYP1A1/p31 plasmid into DH5alpha cells.

9-02-2009

 * Minipreparation of CYP1A1 colonies (six colonies)
 * Test digest of the ligated CYP1A1 construct at 37°C for one hour -> 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 (at 37°C, 1 hour)
 * 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. Design primer with BBB standard, which were adjusted to the plasmid because of the lack of the p52 sequence.
 * 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 (at 37°C, 1 hour)

9-07-2009

 * Digest of promoter with NFkB binding sites (NFkB_prom) with NheI and EcoRI (at 37°C, 1 hour)
 * Digest of half of the volume of the HSP70 promoter with EcoRI and NheI (at 37°C, 1 hour)
 * Digest of half of the volume of the HSP70 promoter with PstI and SpeI (at 37°C, 1 hour)
 * Ligation of the digested NFkB_prom and the HSP70 promoter (EcoRI, NheI) with p31 (EcoRI, NheI; 09/04/09) at 22°C for three hour.
 * Ligation of the HSP70 promoter (EcoRI, NheI) with p31 (EcoRI, NheI; 09/04/09) at 22°C for three hour.
 * Ligation of the HSP70 promoter (SpeI, PstI) with p31 (SpeI, PstI; 09/03/09) at 22°C for three hour.
 * Transformation of the ligated products (above) and the ligated product of CYP1A1 (09/03/09)

9-08-2009

 * Pick some colonies of transformed bacteria (CYP1A1/p31 construct)
 * Miniprep of the CYP1A1/p31 construct.
 * Test digest of the CYP1A1/p31 construct at 37°C for one hour.

9-09-2009

 * Gel electrophoresis image of the test digest:
 * CYP1A1_promoter/p31 construct was included in three positive colonies (Fig. 3). Therefore, this construct is ready for mutagenesis PCR to remove the NheI restriction site at 614 bp.
 * The others have not turned out satisfactory.



9-10-09

 * The sequence of CYP1A1 #2, #8 and #5 are right.
 * Plate out the Addgene plasmids: pJC6-GL3, SBE4-Luc, pGL3-RARE-Luc, pGL3-NFAT-Luc, PUMA-Frag1-Luc, pLDLR-Luc, pHMGCS-Luc

9-11-09

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

9-14-2009

 * PCR of HSP70, NFAT responsive and NFkB responsive promoters.
 * Digest of the PCR products by NheI and EcoRI at 37°C for one hour.
 * Digest of p31_BBB by NheI and EcoRI at 37°C for one hour.
 * 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.

9-15-2009

 * PCR-Purification with HSP70, NFkB and NFAT
 * Gel-extraction of p31_BBB, which was digested by NheI and EcoRI at 37°C for one hour.
 * Ligation: Hsp70 with p31_BBB, NFAT with p31_BBB and NFkB with p31_BBB (at 22°C, 3 hours)
 * PCR troubleshooting with NFAT responsive and NFkB responsive promoters.
 * Amplification of the p52 plasmid (with the HSP70 insert).
 * CYP1A1-muta/p31 PCR product was digested by DpnI (at 37°C, 1 hour).
 * Transformation of the three ligations (above: Hsp70/p31, NFAT/p31 and NFkB/p31) and CYP1A1-muta/p31

9-16-2009

 * Arrival of the sequencing results of the ordered Addgene plasmids.
 * Design of primers for c-Jun promoter, LDL receptor promoter (LDLR), Retinoic Acid Receptor Response Element (RARE) and HMG CoA synthase promoter (HMG CoA)
 * Unfortunaltely, PUMA promoter is not used because of a PstI restrcition site and there is no time for mutagenesis PCR.
 * p52 (with HSP70 insert) is amplified in bacteria (DH5alpha).
 * Miniprep of the following ligation : Hsp70 with p31_BBB, NFAT with p31_BBB and NFkB with p31_BBB
 * The PCR troubleshooting with NFAT and NFkB were analysed by a agarose gel and there were bright bands at ~300 bp, which are a little bit too long. Nevertheless, the inserts could be used for cloning.

9-17-2009

 * Test digest of the ligations p52 (with HSP70 insert), NFAT and NFkB with EcoRI and PstI (37°C, 1 hour -> no right bands!
 * Ligation of NFAT and NFkB (of troubleshooting PCR) with p31 plasmid at 22°C for one hour.
 * No colonies of CYP1A1-muta -> try again the mutagenesis PCR (with special mutagenesis Kit)

9-18-2009

 * Design of primers with BBB standard for the HSP70 insert by analysis of the p52 sequence.
 * Second mutagenesis PCR of CYP1A1
 * Transformation of NFAT and NFkB ligations

9-20-2009

 * pick colonies of NFAT and NFkB

9-21-2009

 * PCR of RARE, LDLR, HMG CoA synthase and c-Jun promoter using primer with BBB_standard. There were two reaction mixture for each promoter. One with Taq polymerase and one with Phu polymerase.
 * Miniprep of NFAT responsive and NFkB responsive promoters and test digest at 37°C for one hour -> no right bands on the agarose gel, so that we skipped this part of the project.

9-22-2009

 * Tranformation of CYP1A1-muta.
 * Analysis of the the following PCR products by agarose gel electrophoresis: RARE, LDLR, HMG CoA synthase and c-Jun promoter (Fig. 4-5).
 * Digest of these PCR products by the restriction enzymes, NheI and EcoRI, at 37°C for one hour.

9-23-2009

 * Gel extraction of RARE, LDLR, HMG CoA synthase and c-Jun insert and combination of the two reaction mixtures (Taq and Phu).
 * Ligation of these extracted inserts with the digested (NheI and EcoRI) plasmid p31 at 22°C for four hours.
 * 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 at 37°C for one hour
 * Transformation of the RARE/p31, LDLR/p31, c-Jun/p31 and HMG CoA promoter/p31 construct.

9-24-2009

 * Ligation of the HSP70 promoter with the digested (NheI and EcoRI) p31 plasmid at 22°C for three hours.
 * Transformation of the HSP70 promoter/p31 construct.
 * Colonies of the RARE/p31, LDLR/p31, c-Jun/p31 and HMG CoA promoter/p31 construct are picked.

9-25-2009

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

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.
 * 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 (Fig. 6-10).
 * Following constructs were send for sequencing:











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.
 * Test digest (NheI and EcoRI) of HSP70 at 37°C for one hour (Fig. 11).
 * Following construct is send for sequencing: HSP70 #9.



9-29-2009

 * Right constructs of HSP70 is: HSP70 #9.

10-12-2009

 * 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: Cell Culture).



Notebook information about the measurement of natural promoters is found in the Notebook Measurement and  Cell Culture.
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