Team:Heidelberg/Notebook synthetic promoters dna

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Revision as of 11:44, 20 October 2009

Week	Days
25	6-15-2009	6-16-2009	6-17-2009	6-18-2009	-	-	-
26	6-22-2009	6-23-2009	6-24-2009	6-25-2009	6-26-2009	-	-
27	6-29-2009	6-30-2009	7-01-2009	7-02-2009	7-03-2009	7-04-2009	7-05-2009
28	7-06-2009	7-07-2009	7-08-2009	7-09-2009	-	-	-
30	-	-	-	7-23-2009	7-24-2009	-	-
31	7-27-2009	7-28-2009	7-29-2009	7-30-2009	7-31-2009	8-01-2009	8-02-2009
32	8-03-2009	8-04-2009	8-05-2009	-	-	-	-
33	-	8-11-2009	8-12-2009	8-13-2009	8-14-2009	8-15-2009	8-16-2009
34	8-17-2009	8-18-2009	8-19-2009	8-20-2009	8-21-2009	-	-
35	8-24-2009	8-25-2009	8-26-2009	8-27-2009	8-28-2009	8-29-2009	-
36	8-31-2009	9-01-2009	9-02-2009	9-03-2009	9-04-2009	-	9-06-2009
37	9-07-2009	9-08-2009	9-09-2009	9-10-2009	-	-	-
38	9-14-2009	9-15-2009	9-16-2009	-	9-18-2009	-	9-20-2009
39	9-21-2009	-	-	-	-	-	-
40	-	-	9-30-2009	-	-	-	-

6-15-2009

Miniprep of GFP template plasmid, pcDNA5/FRT

Nr	pcDNA5/FRT	GFP
1	4,7	39,7
2	5,9	14,9
3	3,7	3,8
4	5,0	7,8

Maxiprep pcDNA/FRT 188.7ng/µL
Extraction of CMV promoter from 2008 distribution
Transformation of DH5a cell with CMV promoter

6-16-2009

DNA synthesis (JeT, cFos, Min)
Purification of cDNA via 2% / 3% agarosegel

=> Only bands at ca. 50 Bp => no successful amplification

Site directed mutagenesis of pcDNA5/FRT and mcherry
Dpn1 digestion
Transformation of DH5a

6-17-2009

No transformations could be observed
Replace Phsuion stocks to Phusion Master Mix from Nathan
Repeat DNA synthesis with a different protocol: 1:10 - 1:1000 dilution of Oligos, 95° 5', 7* [95° 45 58° 45 72° 1'], add 1:10 diluted primers, 95° 5' 25* same temperatures
Annealing successful for JeT and Min

Annealing might not have been successful with Fos beacuse it contains a very repetetive proximal promoter.

[Bild1.jpg|none|frame|Lanes 1-6 Fos (403 Bp); Lanes 7-12 JeT (227 Bp): Lanes 13-18: Min (207 Bp) Lanes 1 : 1µL of 1 :10 diluted oligo 2 : 1 :100 3 : 1 :1000 ;4-6 same, but + DMSO Lanes 7,8,13,14: 1:10 Lanes 9,10,15,16: 1:100 Lanes 11,12,17,18: 1:1000 Even lanes: No DMSO Uneven lanes: DMSO]

gel purfifcation of JeT and Min
Repeat PCR of pcDNA5/FRT and mcherry with lower annealing temperature (58°C) and fresh Polymerase: Follow PCR protcol from Strategene site directed mutagenesis kit.

mcherry PCR might not have been successful because 
we used a template we didn't make ourselves - does it contain TE?

PCR worked for pcDNA5FRT but not for mcherry
DpnI digest of pcDNA5FRT 1,3,4,6

6-18-2009

DNA synthesis for Fos (proximal) and Fos (core)

Left two lanes: Proximal (repetetive), right two lanes (core)

Transformation of DH5a with pcDNA/FRT ΔPstI

Gave many 1000 colonies. Probably a contamination (?), whci hwould explain why there was no mutagenesis (see below)

HeLa cells, MCS7 and U20S were splitted 1:3. Therefore DEMED medium (containing additionally 10% FCS, Pen-Strep, glutamate and non-essential aminoacids) was removed. Cells were washed afterwards with HBSS and trypsinised with 1 ml of trypsin and incubated for 5 min. at 37°C 5% CO2. The used flasks were filled up to a final volume of 7 ml with DMEM. 2 ml of the suspension was transfered to a new flask, 5 ml DMEM was added and incubated for another 3-4 days at 37°C and 5% CO2.

6-22-2009

Split cells (Split again Thursday!)
Prepare 6well-plate with U20S cells for Zeomycine assay
Miniprep pcDNA/FRT ΔPstI
Digest with PstI => No mutation
Use Stratagene QuickChange XL kit for mutagenesis PCR (mcherry, pcDNAand Supercompetent Gold TOP10 cells for transformation => reasonable amount of colonies

6-23-2009

2: pcDNA S-1; 3: pcDNA S-4; 4: pcDNA S-2; 5: pcDNA S-3 6:pcDNA S-5 7: pcDNA S-6 8: pcDNA S-7 9: pcDNA S-8; 10: mCherry D-1 11: mcherry S-2; 12: mcherry S-3; 13: pcDNA D-1 14: pcDNA D-2 15: pcDNA control 16: mcherry control

Added Zeomycine to 6-well platees
Miniprep mutagenisis PCR
Digest with PstI => Mutagenesis successful
PCR to remove EcoRI from pcDNA5/FRT using Phusion polymerase

6-24-2009

DpnI digest
Transformation of Supercompetent Gold TOP10 cells with pcDNA5 ΔEcoRI ΔPstI

6-25-2009

Split cells!

Observation Zeomycine: 0 no killing, 50, 100: significant amount of cells death. 250: ca. 30% of cells death. 500, 750: At least 50% death.
Site directed mutagenesis to remove EcoRI site with stratgene kit, as no colonies had formed previously.

6-26-2009

Change 6well plate medium!

Observation: 0 Zeomycine 100% confluent; 50, 100: some death cells; 250: Many death cells; 500: high confluency (was higher originally), many death cells; 750: most cells death
Transformation with pcDNA5/FRT ΔPstI EcoRI removal PCR product successful (~ 300 colonies)
Miniprep, Test digest with EcoRI

Lane 1: Ladder; Lane 3: Control; Lane 4-12: Clones of site directed mutagenesis to remove EcoRI

6-29-2009

Observation Zeomycine assay: 0 Zeomycine some death, 100% confluent; 50, 100: some remaining cells alive; 250: All cells death but in some areas; 500, 750: All cells death
Abort Zeomycine assay. Rasult: Use 250-500 µg/µl
Maxiprep of pcDNA5/FRT ΔPstI ΔEcoRI
Digest pcDNA5/FRT with PstI and MfeI, digest synthesized promoters with the same enzymes
Digest pcDNA with BclI -> BclI does not cut -> cloning strategy must be changed to use ApaI instead of BclI (new primers designed and ordered)
Gel purification of vector backbone

Lane 2+3: Preparation of Vector backbone after MfeI/PstI digest; Lane 5: plasmid (pcDNA5/FRT ...) Lane 6: BclI digest of plasmid (same behaviour as Lane 5)

Ligation of pcDNA5/FRT ΔPstI ΔEcoRI -MCS with JeT and Min promoters

6-30-2009

Many colonies on all transformations
Pick colonies
Prepare media
PCR of mcherry, GFP

7-01-2009

Miniprep of pcDNA5/FRT ΔPstI ΔEcoRI JeT, pcDNA5/FRT ΔPstI ΔEcoRI Min, pcDNA5/FRT ΔPstI ΔEcoRI (all unmethylated)
Analytic digest with MfeI and PstI to retrieve insert => contaminations and uncut (original, removed insert shows up again), possibly one positive result
Analytic digest of the positive result with SpeI and NdeI to get a larger insert

=> ligations had not worked

mcherry PCR worked, GFP didn't

7-02-2009

Digest pcDNA5/FRT ΔPstI ΔEcoRI from -dam strains with BclI and PstI, Same for mcherry PCR
PCR purification
Ligation
Transformation

7-03-2009

Colony PCR - Colonies 1,2,3,8,10 are promising (to be picked, mini-prepped and test-digested)
Transformation of DH5a with Part BBa_E0040 (GFP)

7-04-2009

Inoculation of LB-Amp media with colony 1,2,3,7,8 and 9
Incubation over night at 37°C

7-05-2009

Miniprep of overnight culture
Digestion of pcDNA5/FRT ΔPstI ΔEcoRI - mCherry ligation with Pst1 (1h 37°C)and Bcl1 (1h 50°C)
HeLa cells, MCS7 and U20S were splitted 1:5 (see above)
6-well plates with the human cell lines were started for transfection (~10^5 cells per well)
Agerose Gelelktrophorese

(lane 1 marker; lane 2 control (undigested); lane 3 colony 1; lane 4 colony 2, lane 5 colony 3; lane 6 colony 7/1; lane 7 colony 7/2; lane 8 colony 8; lane 9 colony 9

7-06-2009

Digest pcDNA5/FRT ΔPstI ΔEcoRI mcherry(PstI-BclI), JeT and Edelman/Min with PstI and MfeI
SAP digest, gel purification of backbones
Ligation
Transformation
Transfect HeLa, U20s and MCS7 cells with pROG4 (??) for stable integration of a FRT site

7-07-2009

HeLa cells, MCS7 and U20S were splitted 1:5 (see above)
Preparation of 6well-plate with HeLa cells, MCS7 and U20S for transfection (stabel integration). Therefore 1*10^5 cells were deluted with DMEM medium to a final volume of 2.5 ml.
Preparation of 8well-plate with Hela cells for transfection (pcDNA5/FRT-mCherry-promoter-ligation). Therefore 1.5*10^4 cells were deluted with DMEM to a final volume of 0,4 ml.
Mini-prep of GFP
PCR of GFP (O. 47 and O.48) (checked via gel)
Inoculation of medium with colonies (pcDNA5/FRT ΔPstI ΔEcoRI mcherry-min-ligation / pcDNA5/FRT ΔPstI ΔEcoRI mcherry-JeT-ligation)

7-08-2009

Digest P.8 (GFPb3mut) PCR (O.47 and O.48) product with Bcl and PstI
Digest P.7 (unmethylated) with BclI and PstI
Ligation
Transformation
Test digest of pcDNA5/FRT ΔPstI ΔEcoRI mcherry-min-ligation / Jet-ligation -> ligation worked (checked by gel)

7-09-2009

Randomized promoter synthesis of HIF-1 using

L1389.O53 HIF-1 a 4µL L1389.O54 HIF-1 b 4µL L1389.O55 Ap1 0,5µL L1389.O56 Random 9 µL L1389.O57 Sp1 0,5 µL L1389.O58 Stop3' 1 µL L1389.O59 Stop5' 1 µL To 200µL; dilute mix 1:10 7 cycles as described for gene synthesis (+/- DMSO), then add primers (O.58, O.59) 1:4 and 1:10 diluted

Digestion of P.9 with ScaI
Transfection with P.9 for stable integration; Transfection with mcherry - Jet/min ligation (transient)

From left to right: 2* no DMSO , 2* DMSO; 1:4 1:10 1:4 1:10

Notice that indeed this is no smear but individual bands. ==

7-23-2009

Retrace and comprehend last weeks' doings
Used pSB1A3 with mCherry (2009 spring distribution plate 1; well 1K) for construction of submission plasmid (add 15 µl water to well and mix)
Transformed DH5α cells once with pSB1A3 and once with plasmid 6 (pcDNA5/FRT ΔPstI ΔEcoRI)

==

7-24-2009

start new cell culture (new media, new cells (HeLa, U2OS from Michaela)...)

  * glutamine and trypsin aliquots are now in freezer II (drawer #3)

pick transformed cells (had single colonies with plasmid 6 as well as with pSB1A3) and culture during the day (5 ml in 37°C incubator with rotation)
synthesize I_pSB1A3 and I_pcDNA from oligos (I_pSB1A3 and I_pcDNA contain BBb standard) twice (first try didn't work)
maxi (250 ml w/ 250 µl Amp and 2,5 ml pre-culture) --> incubate over night

   * those inserts and the according vector will be digested with SspI and PciI (pSB1A3) or SspI and SphI (plasmid 6) respectively

7-27-2009

PCR of EGFP with BclI/PstI primers
Digest P.6 with SspI and SphI, P.23 with SspI and PciI
Digest I_pcDNA with SspI and SphI
Digest I_psb1A3 with SspI and PciI
Ligate
Split cells (HeLa, U2OS --> 1:10)

7-28-2009

Gel electrophoresis of ligation (plasmid 6 + I_pcDNA separate, plasmid 6 + I_pcDNA ligated, pSB1A3 + I_pSB1A3 separated, pSB1A3 + I_pSB1A3 ligated)

[Image:072809_P6_insert_pSB1A3_insert.jpg|none|frame| From left to right: plasmid 6 + I_pcDNA separate, plasmid 6 + I_pcDNA ligated, pSB1A3 + I_pSB1A3 separated (not visible here), pSB1A3 + I_pSB1A3 ligated, 1kb DNA Ladder]

Purification of ligated plasmids with inserts from gel
transformation of the two ligated plasmids (plasmid 6 + I_pcDNA and pSB1A3 + I_pSB1A3), pFRT/lacZeo, mGFPc1, mGFPn2
mGFPc1 and mGFPn2 are two plasmids from Yara containing eGFP
transient transfection of HeLa cells with 3J2, 8J4 (plasmid w/ mCherry and synthetic Jet-Promotor), 8M1, 8M3 (plasmid w/ mCherry and synthetic Min-Promotor), 3, 8 (plasmids with mCherry and CMV-Promotor), positive and negative control (mGFPn1 or plasmid 6 respectively)
Lab Tek 8 chamber allocation:

3J2	8M1	8M3	8J4
3	8	neg	pos

made fresh LB-medium and LB-amp plates

7-29-2009

Transformation of plasmid 6 + I_pcDNA, pSB1A3 + I_pSB1A3 did not work
Gel purification of digested plasmid6 and I_pSB1A3 to delete old Insert
New ligase reaction of plasmid6 + I_pcDNA, pSB1A3 + I_pSB1A3, with higher DNA concentrations
Gel purification of eGFP n1 PCR

Transformation of 8M1, 8M3, 8J2, 8M4 in SCS110 supercompetent cells, and plasmid6 with I_pcDNA, pSB1A3 with I_pSB1A3 in DH5α cells
Plating of Transformed cells

Overnight culture of DH5α with pFRT/lacZeo

sequence of Jet and mCherry in integration plasmid arrived --> mCherry mutagenesis did not work --> only half of mCherry is in the plasmid, therefore no fluorescence after transient transfection

7-30-2009

Over day culture of 8M1, 8M3, 8J4, 3J2 in SCS non-methylating cells
transformation of plasmid6 with I_pcDNA, pSB1A3 with I_pSB1A3 in DH5α cells did not work, maybe mistake in ligation step
synthesis of I_pSB1A3 and I_pCDNA in two parts for each (idea: maybe previously synthesized inserts (I_pSB1A3 and I_pcDNA) were too long --> synthesize two shorter parts, digest them with EcoRI and ligate them)

mutagenesis of mCherry at PstI location
PCR of previously (?) mutagenized mCherry

Digest I_pSB1A3 with SspI, PciI and EcoRI
Digest pSB1A3 with SspI, PciI
Digest I_pcDNA5FRT with SspI, SphI and EcoRI
Digest pcDNA5FRT with SspI, SphI
Digest mcherry PCR, EGFP PCR with PstI, BclI
Miniprep of 8M1, 8M3, 8J4, 3J2
Digest 8M3 and 3J2 with PstI, BclI

Ligate I_x with x and 8M3 / 3J2 with mcherry/GFP

Transformations

cell culture:

transient transfection:
no fluorescence with 8M1, 8M3, 8J2, 8M4 (makes sense, because we had it sequenced and there's only half of mCherry in there)
positive control worked (GFP)
stable transfection:
removed old medium
added fresh medium with Zeocin (stock at 100 mM (used all the stock for 50 ml medium) --> add to medium to get a concentration of 200 µM (1:500))

IDs created

30.7.1 = 3J2 - mcherry fragment + GFP
30.7.2 = 8M3 - mcherry fragment + GFP
30.7.3 = 3J2 - mcherry fragment + mcherry
30.7.5 = BBb integration plasmid (pcDNA5/FRT derived)
30.7.6 = BBb submission plasmid (pSB1A derived)
30.7.7 = 8M3 - mcherry fragment + mcherry

7-31-2009

yesterdays transformations did not seem to work (no colonies on any plates) - at 3pm, colonies were found
Redo Restriction, ligation of 8M3, 3J2 with GFP (31.7.1, 31.7.2)
Transformation of 30.7.5 and 30.7.6 (pSB1A3 + I_pSB1A3, p6 + I_pCDNA)in SCS110 supercompetent cells
PCR of RFP from pSB1A3 (registry spring distribution 2009 plate 1 well 1K) with primers containing BBb (inner restriction sites --> SpeI, NheI)
same for ccdB ("death gene" from registry, spring distribution 2009 plate 1 well 3K)

cell culture:

HeLa and MCF-7 were relatively dense --> splitted 1:10 (so they can be splitted again on Monday and transfection can be prepared)
U2OS were not quite as dense --> splitted 1:6

IDs created

31.7.1 = Ligation of 3J2 + GFP (transformation tbd)
31.7.2 = Ligation of 8M3 + GFP (transformation tbd)

8-01-2009

Transform DH5a with 31.7.1, 31.7.2

8-02-2009

Start Overnight cultures with 30.7.1, 30.7.2, 30.7.3, 30.7.5, 30.7.6, 30.7.7, 31.7.1, 31.7.2

8-03-2009

Miniprep of 30.7.1, 30.7.2, 30.7.3, 30.7.5, 30.7.6, 30.7.7, 31.7.1, 31.7.2
Analytic digest with SphI/PstI for everything but 30.7.5, 30.7.6
Analytic digest with SspI/NheI for 30.7.5, 30.7.6
Gel, sequencing of pcDNA plasmids w/ Jet or Min and mCherry or GFP (test plasmid; also to be transformed tomorrow)

From left to right: 30.7.7#1-4 (used 30.7.7#2 as p.12); 31.7.1#1-4 (used 31.7.1#4 as p.30); 31.7.2#1-2; ladder; control (8M3, 3J2); 31.7.1#3,5-8 (used#6 as p.31); 30.7.1#1-4; 30.7.2#1

From left to right: 30.7.2#2-4; 30.7.3#1-3; ladder; 8m3, 3j2 (control); pcdna5+1#1; psb1a3+I #1,2; pcdna5/FRT (control), psB1a3

insert synthesis for BBb-standardizing of submission and integration plasmids

overnight culture of P.30, P.31 and P.13 (???)

8-04-2009

synthetic promoters

Sequance analysis (alignement to be uploadad)
Maxiprep p12, p30, p31

BBb submission plasmid

Digestion of vectors
Gel purification of vectors
PCR purification of overnight insert ligation
Start overnight ligations

8-05-2009

over night culture (250 ml) of DH5alpha for new competent cells

synthetic promoters

Re-do insertions of Min and JeT with correctly synthesized promoters

BBb submission plasmid

Transformations of ligations

8-11-2009

1. PCR-amplification of Jet promoter with primer 106 and 71 and template p31
2. PCR-amplification of eGFP with primer 97 and 98 and template peGFP-N1
(PCR-amplification of CMV-promoter with primer 109 and 108 and template pGFP-N1 => failed => wrong primers)
(PCR-amplification of CMV-promoter with primer 109 and 110 and template pGFP-N1 => failed => wrong primers)
(PCR-amplification of CMV-promoter + eGFP with primer 109 and 98 and template 3J2 => failed => wrong primers)
3. PCR-amplification of CMV-promoter with primer 109 and 108 and template p6
4. PCR-amplification of CMV-promoter with primer 109 and 110 and template p6
PCR purification of all PCR-product
Restriction digest of peGFP-N1 and Jet PCR-product with AseI and PstI (NE-buffer3 + BSA, 1h at 37°C)
Restriction digest of p7 plasmid and eGFP-PCR-product with BCl1 and Pst1 (NE-buffer3 + BSA, 0,5h at 37°C and 0,5h at 50°C)
Gelelectrophoresis of restricted plasmids and PCR-Products (1% Agerose gel)
Gelextraction of backbones
Ligation of Jet with peGFP-N1-backbone (molar ratio 3:1)
Ligation of eGFP with p7-backbone (molar ratio 3:1)

8-12-2009

PCR-amplification of CMV-promoter with primer 109 and 108 and template pcDNA5/FRT => failed => wrong primers
PCR-amplification of CMV-promoter with primer 109 and 110 and template pcDNA5/FRT => failed => wrong primers
Cloning into p31, Transformation
Pick colonies of the previous day's successful transformations (p.23 + BBB_JeT)

8-13-2009

Minipreped 8 clones of pSB1A3 w/ BBbed Jet
overday culture (5 ml with amp/kana) of 1,3,4,5 from 8-11-2009
Repeat cloning of JeT into pEGFP-N1 using AseI and PstI

8-14-2009

Promotors

minipreped 3-5 (from 8-13-2009)
control digest of 3 (Nhe1, Spe1), 4 (Spe1, Hind3), 5 (Pst1, Sph1), pSB1A3 (Spe1)
gel electrophoresis of control digests and uncut Plasmids 3-5, pSB1A3

8-15-2009

checked transfected cells of flourescence: both transfections with jet/EGFP in pCDNA5/FRT worked (green flourescence), positive controle worked

8-16-2009

picked another 8 colonies from plates 3-5 (see 8-11-09) and started overnight culture

8-17-2009

Synthesized HIF: 2µL LV_HIF-1 a 2µL LV_HIF-1 b 1µL LV_CREB 0.1µL each LV_Ap1 LV_Sp1 1.5µL each LV_Stop3, LV_Stop5; 4.5 µL LV_Random
Synthesized p53: 4µL p530.1µL each LV_Ap1 LV_Sp1 LV_CREB 1.5µL each LV_Stop3, LV_Stop5; 4.5 µL LV_Random
Synthesized negative control: 4.5µL LV_empty 1.5µL each LV_Stop3, LV_Stop5; 4.5 µL LV_Rando
Yielded product in the range of 5kb-200bp
Cut three fragment (long, medium short); PCR purification

8-18-2009

Cloning of cmv core promoter in Jet GFP plasmid (p31)

PCR of cmv core promoter from plasmid 6 whole volume 50 µl: 25 µl Phusion MasterMix, 1 µl 1:4 diluted forward primer, 1 µl 1:4 diluted reverse primer, 0.5 µl p6 plasmid maxiprep as template, 22.5 µl DNase free water.
PCR program: 98 ˚C 30s || 98˚C 10s | 60 ˚C 20s | 72 ˚C 20s || 72 ˚C 5 min | 4 ˚C forever amplied 30 cycles.
PCR product was purified with PCR purification kit and then all digested with HindIII and NheI-HF in Buffer 4 + BSA for 1 hour. Vector (p31) (5 µl maxiprep product) was also digested with HindIII and NheI-HF for 2 hours. for each digestion 0.5 µl enzyme each.
Digestion products were gel purified and ligated with volume ratio of 2:1 (Vector : insert) ligation (10 µl): 2µl HindIII and NheI-HF digested p31 vector, 1 µl

HindIII and NheI-HF digested PCR product (CMV core promoter from p6), 1 µl ligation buffer (Fermentas), 1 µl T4Ligase, 5 µl water. ligation was done at room temperature for 1 hour.

standard transformation in DH5 alpha plated on amp plate.
clony PCR on next day verify the positive colonies.

8-19-2009

PCR of BBbJeT from plasmid P.31 with O.70 and O.71 (correct BBb prefix/suffix oligos)
digested PSB1A3 from registry with EcoRI and PstI
then heat inactivation, SAP digest and gel extraction
digested PCR product of BBbJeT with EcoRI and PstI and subsequent PCR purification
ligation over night at 16°C (ligation product: p.49 --> pSB1A3 with correct BBb standard)

8-20-2009

transformation of ligation (p.49)

8-21-2009

minipreped PSB1A3_BBbJeT (P.49)
test digested with NheI and SpeI
works --> sequenced!

8-24-2009

72 bp missing in PSB1A3 backbone!
sequenced PSB1A3 from registry --> also missing bp --> e-mailed registry people
PCR of P.49 (PSB1A3_BBbJeT) with O.72 and O.87
digested P.6 with SspI and SphI
then heat inactivation, SAP digest and gel extraction
digested PCR product (long insert with all the standard stuff from pSB1A3) also with SspI and SphI
ligation overnight at 16°C (ligation product: p.51 --> standardized pcDNA5/FRT plasmid)

8-25-2009

use BBbJet PCR product (8-19-2009; with complete BBb standard, including NotI restriction site) and digest with EcoRI and PstI; then PCR purification
digested P.31 (pcDNA5/FRT with JeT) with EcoRI and PstI
PCR of CMV (from P.6) with O.108 and O.109

transformed PSB1A3 with ccdB (iGEM 2009 spring submission plate 1 well 3K) into ccdB surviving cells (invitrogen)

8-26-2009

started maxi prep of PSB1A3_ccdB
digested CMV PCR product with NheI and SpeI --> gel purification
digested P.51 with NheI and SpeI --> gel purification

ligated correct BBbJet into P.31 and transformed
ligated BBb_CMV into P.51 and transformed
transformed p.51 (colony 1)

Synthesis of HIF, p53, NFkB responsive promoters; constitutively active promoter; empty/random promoter:

HIF	NFkB	p53	constitutive	empty
2,5µL HIF-1 (O.53) 2,5µL HIF-2 (O.54) 1µL CREB(O.89) 3µL HIF-rev (O.189) 3µL Random (O.56) 1µL Stop 5 new (O.187) 1µL Stop 3 (O.58) .2µL each Ap1, Sp1 (O.55, O.57)	2,5µL NFkB-1 (O.93) 2,5µL NFkB-2 (O.94) 3µL NFkB-rev (O.194) 3µL Random (O.56) 1µL Stop 5 new (O.187) 1µL Stop 3 (O.58) .2µL each Ap1, Sp1, CREB, NFY (O.55, O.57, O.89, O.90)	5µL p53 (O.91) 3µL p53 rev (O.188) 3µL Random (O.56) 1µL Stop 5 new (O.187) 1µL Stop 3 (O.58) .2µL each Ap1, Sp1, CREB, NFY (O.55, O.57, O.89, O.90)	2µL Sp1 (O.57) 1µL Ap1 (O.55) 1µL CREB (O.89) 0.5+0.5µL NFkB (O.93/94) 4µL Random (O.56) 1µL NFkB rev (O.194) 1µL Stop 5 new (O.187) 1µL Stop 3 (O.58)	5µL Random (O.56) 5µL Empty (O.95) 1µL Stop 5 new (O.187) 1µL Stop 3 (O.58)

Run Gene synthesis PCR (compare material and methods)
Gel purify: Exclude everything shorter than 100 bp. Excise two fragments: Everything < 750bp, and everything >750bp

Preparative (2% agarose) gel of random promoter synthesis. PCR product length is spread between >6000 and <100 bp.

8-27-2009

PCR of mCherry from p.41_muta with O.50 and O.71
digested PCR product with AvrII and BclI and gel purified product (to be the insert)
digested p.7 with NheI and BclI --> looks strange (three bands) --> redo
digested p.7 with NheI, then PCR purification

PCR of p.29 (pSB1A3 with P1010) and p.23 (pSB1A3 with J04450) with O.176/O.177 or O.174/O.175 respectively
- digested PCR products with EcoRI and PstI
- gel purification of pcr product
digested p. 49 with EcoRI and PstI
- gel extraction of plasmid backbone of p.49 (pSB1A3 with BBb-standard flanking regions)

Synthesis of HIF, p53, NFkB responsive promoters; constitutively active promoter; empty/random promoter:

Cut gel extracted promoters with SpeI/HindIII, Cut p.31 with SpeI/HinDIII. Use a long digestion time (1,5h + 1h SAP)
Gel purify vector, PCR purify inserts
Ligation: 2,5h @ 20°C
Transformation

--- Protocol optimization

In order to find out the otpimal conditions: Run a gradient PCR for HIF and const. synthesis (using oligo concentration as specified on 8-26-2009):

	1	2	3	4	5
Const, PCR purification after 1st 7 cycles	Sample 1: 57°C 1st annealing 58°C 2nd annealing	2: 58,36°C 1st 61,82°C 2nd	3: 59,27°C 1st 64,36°C 2nd	4: 60,64°C 1st 68,18°C 2nd	5: 62°C 1st 72°C 2nd
HIF, PCR purification after 7 cycles	Sample 6: 57°C 1st annealing 58°C 2nd annealing	7: 58,36°C 1st 61,82°C 2nd	8: 59,27°C 1st 64,36°C 2nd	9: 60,64°C 1st 68,18°C 2nd	10: 62°C 1st 72°C 2nd
Const, no PCR purification after 7 cycles	Sample 11: 57°C 1st annealing 58°C 2nd annealing	12: 58,36°C 1st 61,82°C 2nd	13: 59,27°C 1st 64,36°C 2nd	14: 60,64°C 1st 68,18°C 2nd	15: 62°C 1st 72°C 2nd
HIF, no PCR purification after 7 cycles	Sample 16: 57°C 1st annealing 58°C 2nd annealing	17: 58,36°C 1st 61,82°C 2nd	18: 59,27°C 1st 64,36°C 2nd	19: 60,64°C 1st 68,18°C 2nd	20: 62°C 1st 72°C 2nd

8-28-2009

Protocol optimization

Gel extraction of yesterday's gradient PCRs (one fragment per lane, include everything > 100 Bp)

Upper Gel:Lane 1-5: Sample 1-5; Lane 8-10: Sample 8,9,10 (Sample 6 and 7 were lost); lane 11: Sample 11
Lower Gel: Lane 1-4: Sample 12-15; Lane 5-8: Sample 16-20

miniprep of P.51 (colony 1), P.51 with BBb_CMV and P.31 with complete BBb_Jet
test digested P.31 with complete BBb_Jet with NotI
test digested P. 51 and P.51 with BBb_CMV with NheI and SpeI

digested P.7 that was already digested with NheI with BclI (50°C) for 2,5 hours, because it didn't work well yesterday --> put on gel

Synthesis of HIF, p53, NFkB responsive promoters; constitutively active promoter; empty/random promoter:

Pick 25 colonies each from plates containing putative long and short p53,NFkB,HIF promoters; Pick 15 colonies each from plates containing long and short putative constitutive promoters; Pick 6 colonies each from plates containing long and short random promoters.

important plasmids (finished):
- p.10: pcDNA5/FRT/eGFP (with original CMV = positive control for transfection)
- p.31: pcDNA5/FRT/eGFP_BBbJeT (complete BBb standard now)
- p.48: pcDNA5/FRT/eGFP_BBbJeT_CMV (incomplete BBb standard)
- p.49: pSB1A2_BBbJeT
- p.51: pcDNA5/FRT_BBbJeT (with standard flanking regions from pSB1A2)
- p.53: pcDNA5/FRT_BBbCMV

8-29-2009

Synthesis of HIF, p53, NFkB responsive promoters; constitutively active promoter; empty/random promoter:

Miniprepped the following colonies:
- HIF S 1-24
- HIF L 1-24
- NFkB S 1-24
- NFkB L 1-24
- p53 S 1-24
- p53 L 1-24
- Const S 1-12
- Const L 1-12
- Empty S 1-4
- Empty L 1-6

8-31-2009

Synthesis of HIF, p53, NFkB responsive promoters; constitutively active promoter; empty/random promoter:

Test digest, nanodropping

Upper Gel:HIF S Lower Gel: HIF L	Upper Gel:NFkB S Lower Gel: NFkB L
Upper Gel:const S, ladder, const L Lower Gel: Empty S 1-4, Empty L 1-6, p53 1-16	p53 S 17-24 and p53 L

Picked DNAs to be transfected and diluted to 33,33 ng/µL:
- HIF S 3-20,23,24
- HIF L 4,6-9,11,16,17,19,20,21,23
- NFkB S 2,4,5,7,15-18,23
- NFkB L 9,14,16,22-24
- p53 all
- const S 4,5,10,12
- const L 1,4,5
- empty all

Transfections

9-01-2009

Synthesis of HIF, p53, NFkB responsive promoters; constitutively active promoter; empty/random promoter:

Induction and prelimary screening
- HIF was induced by hypoxia bag for 1,5 h
- p53 was induced by topoisomerase inhibitor
- NFkB was induced by TNF-a

Protocol optimization

Cloned optimization samples 1-5 into p31 (ligations, transformations)
Tried the following PCR protocol for HIF (same mixture as above) in order to increase construct length (Sample thus generated is referred to as Sample W below):


Initiale denaturation	95 °C, 5 min	1 cycle

denaturation/annealing	95 - 58 °C decrement step, 15 sec per degree Celsius
annealing	58 °C, 20 sec
extension	72 °C, 1 minute	7 cycles

	4 °C	forever
	PCR purification
	25 cycles as normal (69°C annealing temperature)

9-02-2009

Synthesis of HIF, p53, NFkB responsive promoters; constitutively active promoter; empty/random promoter: Screening results

All constitutive promoters were active
Random promoters were non-active
Numerical evaluation of regulated (HIF, NFkB, p53) promoters: From 0 = no fluorescnence to 3 = strong fluorescence can be found in Results 9-1-2009
- HIF: Induction time was too short
- p53: Most cells were death, as the cells were left in camptothecine for too long
Sequence analysis shows that the PCR protocol ("ROE-PCR") works (see images below), leaving the following remarks
- Long consesus motifs (e.g. p53) must be added in a higher proportion
- Obviously, small variations in pipetted oligo volume critically affects primer length annd composition (No other explanation for NFkB "promoters" being so short, compare Gel image and sequencing results).

Sequence of constitutive promoters.	Sequence of HIF responsive promoters
Sequence of NFkB responsive promoters. Note that samples were too short, NFkB was re-synthesized.	Sequence of p53 responsive promoters. Note that p53 binding motifs are not present in the desired quantities. Therefore, p53 was re-synthesized.

This is the way forward

Measure Const L 4, Const L 5, Const S 10, Const S 4 (+ Const L1, S5) → Four constitutive promoters of different strength and of our own making!)
Transfect again:
- HIF S 3-20,23,24 ; HIF L 4,6-9,11,16,17,19,20,21,23 → Because bad choice of timepoint: Wait ~5h
- p53 S 8, 13, 22, 23, 24; p53 L 1, 3, 9, 11, 13, 17, 18, 19 → look promising, but bad choice of timepoint: Wait 30, 45, 60 minutes; alos induce by using UV light.
2nd round of promoter synjthesis
- SREBP
- AHR
- pPARgamma
Improving existing protocols
- p53 (higher content of p53 oligos)
- NfkB again (less start and stop?)

9-03-2009

New attempt to synthesize NFkB and p53 responsive promoters:

Gene synthesis (compare Material and Methods) using the following oligo concentrations and a PCR purification step after the 1st seven cycles.

p53	NFkB I	NFkB II	Activator Mix
6µL p53 (O.91) 5µL p53 reverse (O.188) 1µL random (O.56) 1µL Activator Mix 0.8µL Stop 5 new (O.187) 0.8µL Stop 3 (O.58)	3µL NFkB-1 (O.93) 3µL NFkB-2 (O.94) 5µL NFkB-rev (O.194) 1µL Random (O.56) 1µL Activator Mix 0.5µL Stop 5 new (O.187) 0.5µL Stop 3 (O.58)	3µL NFkB-1 (O.93) 3µL NFkB-2 (O.94) 4µL NFkB-rev (O.194) 3µL Random (O.56) 2µL Activator Mix 0.8µL Stop 5 new (O.187) 0.8µL Stop 3 (O.58)	2µL each Ap1, Sp1, CREB (O.55, O.57, O.89) 1µL each NFY, Empty (O.90, O.95) Water to 30µL

Gel extraction. Excised two fragments per lane, one >1000 (L) and one <1000 and >150-200 (S)

Synthesis of p53, NFkB (I and II) promoters
1% agarose gel. Note that less short products exist due to lower concentrations of Stop 5 and Stop 3 oligos.

9-04-2009

New attempt to synthesize NFkB and p53 responsive promoters:

Digest p31 with HinDIII, SpeI
Digest yesterday's gel extractions (p53 S, p53 L, NFkB I S, NFkB I L, NFkB II S, NFkB II L) with HindIII, SpeI
Ligation
Transformation

Synthesis of HIF, p53 promoters from the first synthesis round:

Screening of p53 again, 40 minutes after induction:
- Clone p53 L 17 and p63 L 19 show induction; they showed induction in the 1st screening also and will be transfected for FACS measurement
Screening of HIF: HeLa Cell line not suited, dies too fast in minimal media -> to be repeated in MCF-7 cells

9-06-2009

New attempt to synthesize NFkB and p53 responsive promoters:

Picked colonies for miniprepping:
- NFkB I S 6*
- NFkB I L 12*
- NFkB II S 12*
- NFkB II L 12*
- p53 S 12*
- p53 L 18*

9-07-2009

New attempt to synthesize NFkB and p53 responsive promoters:

Minipreps
Test digests

Synthesis of p53, NFkB (I and II) promoters - test digest
1% agarose gel. Note that avg. product length is longer than in the previous synthesis round due to lower start/stop concentrations.

9-08-2009

New attempt to synthesize NFkB and p53 responsive promoters:

Transfections (+/- TNF-a for NFkB, +/- xxx for p53)

9-09-2009

Synthesizing AHR, SREBP and pPARy

Gene synthesis (compare Material and Methods) using the following oligo concentrations and a PCR purification step after the 1st seven cycles.

SREBP	AHR	pPARγ	Activator Mix
5µL SREBP (O.208) 4µL SREBP reverse (O.209) 1µL Sp1 (O.57) 2µL random (O.56) 1µL Activator Mix 0.8µL Stop 5 new (O.187) 0.8µL Stop 3 (O.58)	5µL AHR (O.212) 4µL AHR reverse (O.213) 2µL random (O.56) 1,5µL Activator Mix 0.8µL Stop 5 new (O.187) 0.8µL Stop 3 (O.58)	5µL pPARγ (O.210) 4µL pPARγ reverse (O.211) 2µL random (O.56) 1,5µL Activator Mix 0.8µL Stop 5 new (O.187) 0.8µL Stop 3 (O.58)	2µL each Ap1, Sp1, CREB (O.55, O.57, O.89) 1µL each NFY, Empty (O.90, O.95) Water to 30µL

Gel extraction. Excised two fragments per lane, one >1000 (L) and one <1000 and >150-200 (S)
Digest p.31, p48 with HinDIII, SpeI for 4h; 1h + SAP; Gel extraction
Digest Gene synthesis products with HindIII, SpeI
Ligation

Screening of NfKb, p53 responsive promoters (of second synthesis round)

NFkB was transfected into U2OS and induced by addition of 2,5µM TNF-α
p53 was transfected into MCF-7 and induced by addition of Camptothecin (CPT), a topoisomerase inhibitor
Screening by eye / fluorescence microscopy
NFkB: Promising clones. Screening will be repeated, including a second control (dye-free DMEM) as NFkB might be induced by starvation
p53: Obviously, starvation did induce p53 whereas Camptothecin did not (maybe cells were harmed too much by the time p53 would have been active). Starvation does induce p53 as p53 is a target of AMP-Kinase. We will repeat the screening with a p53-inhibitor.

Screening of putative NFkB regulated promoters. 1-6 corresponds to relative GFP expression strength levels.

Screening of putative p53 regulated promoters. 1-6 corresponds to relative GFP expression strength levels.

9-10-09

Transformation

9-14-09

Minipreps
Test digest to find only AHR synthesis is correct (pictures tbd)

9-15-09

Induction of U2OS cells transfected with putative NFkB responsive promoters
Screening by TECAN for NFkB of second synthesis round: Results show (a) that TECAN (despite initial advise by our favourite advisor telling us to screen by eye, instead of TECAN) and (b) many promoters show regulation. These will have to be measured in detail by flow cytometry. Also, for future TECAN screenings, a timepoint zero measurement will have to be added, but this series of measurement still is acceptable as comparable initial fluorescence levels were found by eye/microscopy as for previous screenings (data not shown).

TECAN measurement results. A corning 96 well plate (transparent, flat) was used. Promoters upregulated by TNF-a are marked greeen, one promoter appears to be down-regulated (marked blue).

Repeat promoter synthesis for AHR, pPARγ, SREBP and Estrogen Receptor

9-16-09

Ligation, Transformation of repeated nuclear receptor promoter synthesis

9-18-09

Screening pf p53, HIF
p53 screening showed: some promoters are downregulated under p53 induction, which is not how JeT (p31) behaves.

Screening of putative p53-regulated promoters by TECAN. the third table compares the values of the 1st with the values of the 2nd table. Promoters marked bold were selected for further characterization by flow cytometry.
p53 screening showed: some promoters are downregulated under p53 induction, which is not how JeT (p31) behaves.

Screening of putative HIF-regulated promoters by TECAN. the third table compares the values of the 1st with the values of the 2nd table. Promoters marked bold were selected for further characterization by flow cytometry.

9-20-09

Minipreps of second synthesis round for Estrogen Receptor, pPARγ and SREBP-regulated promoters

Test digest of second synthesis round for nuclear recptor regulated promoters 1% agarose gel. Digest with HindIII and SpeI, 1h

9-21-09

Synthesis was remade for the Promoters stated above. We improved gel extraction quality. Still, quality synthesis remained low. We selected clones from the gel that were subjected to screening subsequently. Later, we found out that probably damaged HinDIII enzyme was responsible for the bad synthesis quality.

9-30-09

Screenings for AHR, Estrogen Receptor, pPARγ and SREBP-regulated promoters were finished in the previous days (compare Cell culture Notebook).
For all indcution times, srugs and cell lines, compare Material and methods
AHR results, first round of screening (with TCCD for inducion)

putative AHR responsive promoters, screening by TECAN

AHR results, second round of screening (with Benzo[a]pyrene for induction)

putative AHR responsive promoters, screening by TECAN

pPARγ results

putative pPARγ responsive promoters, screening by TECAN

Estrogen Receptor results, first round of screening

putative pPARγ responsive promoters, screening by TECAN

October 2009

In the weeks before wiki freeze, selected promoters were characterized by the measurement subgroup (see their Notebook ). At the same time, further screenings were conducted as described Also, some promoters were characterized roughly by triple TECAN-reads (namely, pPARγ and p53 responsive clones), or attempted to characterize in this was. We provide all spreadsheets thus generated in a two zip file: raw data and analyzed data. All Spreadsheets are commented with date of measurement. The only of those characterization which yielded reliable results we decided to publish on the registry is for pPARγ (see below or in analyzed data).

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Team:Heidelberg/Notebook synthetic promoters dna

From 2009.igem.org

Revision as of 11:44, 20 October 2009

Contents

6-15-2009

6-16-2009

6-17-2009

6-18-2009

6-22-2009

6-23-2009

6-24-2009

6-25-2009

6-26-2009

6-29-2009

6-30-2009

7-01-2009

7-02-2009

7-03-2009

7-04-2009

7-05-2009

7-06-2009

7-07-2009

7-08-2009

7-09-2009

7-23-2009

7-24-2009

7-27-2009

7-28-2009

7-29-2009

7-30-2009

7-31-2009

8-01-2009

8-02-2009

8-03-2009

8-04-2009

8-05-2009

8-11-2009

8-12-2009

8-13-2009

8-14-2009

8-15-2009

8-16-2009

8-17-2009

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

9-06-2009

9-07-2009

9-08-2009

9-09-2009

9-10-09

9-14-09

9-15-09

9-16-09

9-18-09

9-20-09

9-21-09

9-30-09

October 2009