Team:Freiburg bioware/Notebook/October

01.10.09, Hannes, Manu, Laura, Gerrit, Sarah, Christoph, Caro, Julia, Anika, Isabel

•  Tetracycline; 10 x 1ml, Concentration: 25 g/l, stored in -20°C, box with antibiotics
•  Chloramphenicol; 10 x 1ml, Concentration: 25 g/l, stored in -20°C, box with antibiotics
•  plasmid preparation of pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4
•  plasmid preparation of pEX-Strep-Dig-Split-Fok(active) Klon 1
•  glycerin stock of pEx-His-FluA-Split-Fok(inactive)-RBS-Strep-Dig-Split-Fok(active) Klon 4 (RV308), stored at -80°C

• protein purification of HisFluASplitFoki (expressed in BL21de3) with NiNTA column
• SDS gel of protein purification HisFluASplitFoki [[Image:Freiburg09_011009_pg_HisFluASplitFoki011.jpg|none|thumb|SDS-Gel; HisFluASplitFoki; Lanes: NEB prestained protein marker, elution fraction 1, elution fraction 2, elution fraction 3, elution fraction 4, elution fraction 5, elution fraction 6, elution fraction 7, flow through fraction 2, washing fraction 2|400x400px]]
• pooled fraction 2-5, dialysis over night in dialysis buffer (30mM NaCl, 20mM Tris-HCl, pH 7.4)
• phage display: - desalt ligation products (for electroporation)

•  Overnight Culture RV308 for Competent Cells, on shaker in 37 °C room
• analysis of sequences from 28.09.09
• digest of pExStrepDigSplitFoka prep from 30.09.09 Plasmid: 15 µl

• digest of PCR product RBSStrepDigSplitFoka from 30.09.09 Plasmid: 10 µl

Agarose gel; Lanes: 1.Gene ruler ladder mix of fermentas, 2.pExHisFluASplitFoki, 3.pExStrepDigSplitFoka, 4. RBS_StrepDigSplitFoka

• Inoculation of - pJS419_StrepDigSplitFoka

• digest pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning
• Inoculation of pEx_HisFluASplitFoki in BL21de3 with colony of transformation from 28.09.09 --> make glycerol stocks tomorrow
• agarose gel (1%) of the digest of pMA_ShortlinkerFoki/a, pMA_MiddleLinkerFoki/a and pMA_scFa-anti_NIP for recloning
• cut out insert bands (digestion of pEX didn't work, because there's no EcoRI recognition site any more)
• gel slices stored at -4°C over night

02.10.09 Laura, Manu, Anika, Julia, Hannes, Gerrit, Isabell, Sarah, Caro, Timo, Christoph, Max

• phage display - OD600 of the pre-culture should be 0,2

• glycerol stock of pEx_HisFluASplitFoki in BL21de3
• SDS gel of pool (elution fraction 2-5) from protein expression (HisFluASplitFoki) from 01.10.09 --> do a Western Blot
• new ampicilin 100 aliquots
• preparation of M13dsDNA
• chem. competent cells aliqots
• Periplasma Project Digestion:

•  Gelextraction Min Elute Gel Extraction Kit

• Ligation - with Quickligase JS119-StrepFokA

• Transformation - 2YT-Medium 950 µL to 50µl cells and DNA

•  Preparation of overnight cultures 5 ml LB medium each - pBad+Kan

• Amp

• Digest: - pMASplitFoka

• preparative gel [[Image:Freiburg09_021009_umklonierung.JPG|none|thumb|Agarose gel; Lanes: pMASplitFoka, 2.pMAFoka, 3.pMAFoki, 4.pMASplitFoki, 5.pMALongLiFoka, 6.pMALongliFoki, 7.pMAShortli, 8.pMAMiddleli, 9.pMALongli, 10.pExStrepFluA<|400x400px]] ->interpretation: just the linker resulted in too short fragments, thus the hybridized linkers have to be cloned in pMA directly
• gelextraction of digest from 02.10.09 and also pMAShortliFoka, pMAShortliFoki, pMAMiddleliFoka, pMAMiddleliFoki from 01.10.09
• digest and ligation into new pMA:

•  Transformation of:

• plasmidprep of:

• testdigest of: V1. pEX_DsbA+Strep+Dig+Split+FokA Clone1 Quick

03.10.09 Manu, Hannes, Gerrit, Caro, Timo, Christoph

• plasmid prep of pET and pBad and Glytsocks(Xbl)
• new aliquots Ampicilin (70%EtOH)
• Test digest M13dsDNA+fokI as control for dsDNA , the pictures showed the two expected lanes(hardly visible on the printout). [[Image:Freiburg09 091003 M13test 005.jpg|none|thumb|Agarose gel; M13dsDNA test digest; Lanes: DNA ladder mix ,empty , M13dsDNA02.10.09, M13dsDNA02.10.09 digest fokI |400x400px]]

• digest of - pEx-Strep-Dig-LongLinker-Fok(inactive)

• digest of - pMA with XbaI and AgeI

• Ligation of: - Strep-Dig-LongLinker-Fok(inactive)

• inoculation of: -pMASplitFokA clone1 19.08.09

• inoculation and plasmid preperation: -pEX_strepFluA
• Quenching test with HisFluASplitFoki and fluorescin-tagged oligos
• BB Ago Pcr via Taq
• Started makin new VCS M13 Phages see Protocol day 1

Plan for 04.10.09

• inoculation of - pMA-Strep-Dig-LongLinker-Fok(inactive)

• digestion of pEx-Strep-Flua with AgeI and PstI
• digestion of pMA-Short/Middle/Long/Split-Fok(active and inactive)
• ligation of the above parts and transformation into RV308
• plasmid preparation of... -pMASplitFokA

04.10.09, Laura, Anika, Max

•  Plasmid preparation of -pMASplitFokA

•  Glycerolstock of

•  Pellets of

• Digest of 1. pEXHisDigMiddleLFoki 2. pMaScFaantiNIP 3. prep of Ligation pEX+CAT of 09.07.09 old 4. pMAShortLFoka

• Preparative Gel of Digest

Agarose gel; Lanes: pMASplitFoka, 2.pMAFoka, 3.pMAFoki, 4.pMASplitFoki, 5.pMALongLiFoka, 6.pMALongliFoki, 7.pMAShortli, 8.pMAMiddleli, 9.pMALongli, 10.pExStrepFluA

• Gelextraction
• Ligation of

• Dephosphorylation of pBAD Vector:

• Transformation of

• test digest of plasmid preps from today -> run on a gel with digest pET39, xbaI, ecoRI from Tobi
• starter culture of pEx_DsbA_HisDigSplitFoka -> modified expression has to be done tomorrow
• inoculation of pJS418 and pJS419 for glycerolstocks tomorrow
• Taq AGO BB PCR did not work
• Phageproduction day 2

05.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes, Gerrit, Anika

•  3l LB and 2L LB Agar
• Periplasma Project
• digest pExHisFluASplitFoki (prep from 24.08.09) and RBS_StrepDigSplitFoka (prep from 29.09.09)
• test digests of pMAFoka, pMALongliFoki, pMAFoka old, pMAFoki old, pExHisFluASplitFokiStrepDigSplitFoka
• 1) Gelextraction

• 2)Ligation

• 3)Transformation

• '''Mini prep with with Qiagen Spin Miniprep Kit of''' - pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3

• Made Glycerolstocks of
• 700µl Cellsuspension+ 300µl Glycerol - pMA-Strep-Dig-LongLinker-Fok(inactive)1,2,3

• '''Test Digestion of'''
• put in each sample: 5µl DNA, 2,5µl H2O, 1µl Buffer 3, 1µl EcoRI, 1µl PstI, 1µl BSA - pMA-Strep-Dig-LongLinker-Fok(inactive)3

•  Transformation of

• Expressionsculture of pEx_DsbA_HisDigSplitFoka in 6X2l flasks in 600ml DYT medium each - induced with 0,7mM IPTG and took samples T0-T5

• PET 39 b+ ssDNA "PCR" with new template from digestion of yesterday -> very few product, as well as non specific ones
• PET 39 b+ ssDNA "PCR" with more cycles and higher annealing temperature

06.10.09, Laura, Manu, Julia, Christophe, Caro, Hannes, Gerrit, Anika

• digest - pBAD vector

• testdigest of pExHisFluASplitFoki_StrepDigSplitFoka DNA: 5µl

• preparative gels of digests

preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pBADvector, 3. pBADinsert/dummy, 4.pJS418	preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka

• testdigest of pMAdsba DNA: 5µl

• His-tag purification of HisDigSplitFoka (periplasm export) with Ni-NTA column. Washing buffer: 25mM imidazole -cells were sonicated for 2 x 1min before filtering with 0.45µm and 0.22µm filter

• Ligation - Dephphorylation of pBAD Gelex

•  Insert (Dummy)

•  Transformation --> pBAD
•  Insert (Dummy)

• Ligation and Transformation of

• Sequencing: 27µl H2O; 3µl DNA -pMA Dbsa clone 2

• Inoculation of ER2738 M13: 250ml Erlenmeyerkolben+ 50ml LB+TET in 37°c room overnight
• Yesterdays improved pcr resulted in a lot of product, but still unspecific ones.. made new one with even higher annealing temperature (69°C) and a bit less cycles (35)
• New AGO M13 ssDNA assay using 5`-phosphorylated Oligos A1 and A4 _see gele_ no differend results were obtained compaired to the assays using unphorphorylated oligos
• Started dialysis to transfer the leftover AGO-proteins into the assay buffer

FokI wildtype cutting
Hybridize M13 ssDNA with Fok control oligos 2 and 3

Volume	Reagent
10 µl	M13 ssDNA (c = 127 ng/µl, 17.08.09, sample #5.2)
5 µl	MgCl2 (5 mM)
5 µl	Tris-HCl pH 8 (100 mM)
2 µl	Fok control 2 (10 ng/µl; 1 µM)
2 µl	Fok control 3 (10 ng/µl; 1 µM)
30 µl

The hybridization was also done with just Fok control 2 and with no oligos at all.
Incubation with the thermocycler and the program ORIGAMI0 (heat to 95°C and cool down slowly to 37°C over 1 hour).
10 µl of the hybridized DNA were used for a cutting experiment with 1,2 µl buffer 4 (NEB) and 0.5 µl FokI (wildtype Fok from NEB). The digest was incubated for 30 minutes at 37°C.

Agarose gel of FokI cutting M13 ssDNA

Agarose gel of FokI cutting M13 ssDNA

Lane 1: GeneRuler DNA Ladder Mix (Fermentas, 6 µl);
Lane 2: Untreated M13 ssDNA (250 ng);
Lane 3: Digest of M13 ssDNA with FokI and without oligo;
Lane 4: Digest of M13 ssDNA with FokI and with one oligo;
Lane 5: Digest of M13 ssDNA with FokI and two oligos.

07.10.09 Laura,Christoph, Hannes, Timo, Julia, Caro, Anika

• SDS gel of protein purification of HisDigSplitFoka (periplasm) from 06.10.09 [[Image:Freiburg09_071009_pg_DsbAHisDigSplitFokA_28grad006.jpg|none|thumb|SDS gel, pEx_DsbA_HisDigSplitFoka, lanes: NEB prestained protein marker broad range, Elution fraction 1, Elution fraction 2, Elution fraction 3, Elution fraction 4, Elution fraction 4, Elution fraction 5, Elution fraction 6, flow through fraction 3, washing fraction 2, periplasm extract (frozen over night at -80°C)|400x400px]]

•  Inoculation 4 Clones respectively:

•  Starter culture of pEx-DsbAHisDigSplitFoka in Bl21de3
• two step PCR assembly of DsbA, His_Fos, and SplitFoka - program name: Assembly

• preparative gel of the PCR samples -> primer haven't been diluted an probably made all secondary structures, has to be repeated
• digest of pBAD with AgeI and new PCR with insert digested with XmaI
• preparative gel with digest and
• test digest of pJS419_HisDigSplitFoka and pJS419_StrepDigSplitFoka DNA: 5µl

• plasmidpreparation of - pEXHisFluASplitFoki

• digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka pExHisFluASplitFoki: 15µl

•  Gelextraction of digest 1) Insert: pEX-Strep-Dig-Split-Foka( digested with xba and pst)

• preparative gels of digests

[[Image:|none|thumb|preparative agarose gel, lanes: 1. Insert2. pEX-Vector, 3. PCR- RBS Product]]

[[Image: |none|thumb|preparative agarose gel, lanes: 1. Genruler DNA ladder mix of fermentas, 2. pJS419, 3. pExHisSplitFoka, 4. pExStrepDigSplitFoka |300x300px]]

•  Ligation - 6µl Insert

• Transformation - All in XL1blue

• Stardet production of Phages baering the phagmid vektors with pJS_TorA-flag-AGO-noAmber-CDg3p and the pJS_TorA-flag-AGO-CDg3p, 480 ml each. see phageproduction protocoll day 1
• prepaired ELISA with anti-flag antibodies
• made electro competent cells for transformation with the ago phagmidbibliothek

08.10.09 Manu, Christoph, Julia, Caro, Timo, Hannes

• Poured SDS-Gels
• Phage ss DNA -Over night cultured ER2738 in 100ml LB+Tet dilute on OD600=0,4 in 50ml LB+Tet

• Miniprep - 2 clones respectively:

• Finished Phage production(see protocol day 2): We obtained approximately 3.8
• 10^12 pJS_TorA-flag-AGO-noAmber-CDg3p (449) and 1.1
• 10^13 pJS_TorA-flag-AGO-CDg3p (448) -> aplyed all of them to the anti-Flag ELISA

• Anti-Flag ELISA was successfull with a slight but detctable signal via anti M13 VCS Antibodies (with peroxidase):

	1	2	3	4	5	6	7	8	9	10	11	12
A	0.0470	0.0420	0.0440	0.0430	0.0480	0.0510	0.0460	0.0470	0.0470	0.0480	0.0450	0.0460
B	0.0470	0.0460	0.0480	0.0480	0.0460	0.0450	0.0470	0.0560	0.0470	0.0450	0.0450	0.0440
C	0.0460	0.0470	0.0500	0.0450	0.0430	0.0460	0.0450	0.0460	0.0430	0.0420	0.0440	0.0470
D	0.0490	0.0470	0.1590	0.1610	0.1920	0.1720	0.2210	0.2500	0.2130	0.2680	0.0460	0.0480
E	0.0490	0.0490	0.0450	0.0490	0.0430	0.0490	0.0460	0.0430	0.0460	0.0420	0.0450	0.0440
F	0.0490	0.0430	0.0470	0.0440	0.0470	0.0470	0.0440	0.0450	0.0500	0.0460	0.0450	0.0450
G	0.0500	0.0440	0.2530	0.2510	3.9180	3.8800	0.9560	0.9380	1.2420	1.2360	0.0440	0.0460
H	0.0480	0.0430	0.0510	0.0470	0.0480	0.0460	0.0470	0.0450	0.0450	0.0440	0.0460	0.0460

•  Made new PCR for ssDNA from pET39b+ fragment and gained approximately 200 ng of ssDNA after PCR and gelextraction
• digest of pExHisFluASplitFoki, pExStrepDigSplitFoka and RBS_StrepDigSplitFoka pExHisFluASplitFoki: 15µl

•  Gelextraction of digest 1) Insert: His-Dig-Split-Foka( digested with xba and pst)

• preparative gels of digests ->see picture

• ligation of -pEX-His-Flua-Split-Foki_His-Dig-Split-Foka

• transformation of ligations
• inoculation of pJS419_HisDigSplitFoka in LB + chloramphenicol
• new two step PCR with all three plasmids for Fos construct (pMADsbA, pMAFos, pMASplitFoka, this time with right primer dilution and preparative agarose gel ->interpretation: no band of 921bp was visible

• one step PCR of pMADsbA, pMAFos, pMASplitFoka separated and preparative agarose gel ->see picture

• new one step PCR of pMADsba with newly prepared 1:1000 dilution
• test digest of plasmidpreparations from today ->see picture

09.10.09 Manu, Julia, Caro, Laura, Christopherus, Hannes, Timo, Max, Anika

• Phage ss DNA

• Miniprep - pJS 419-his-dig-split-foka (clon 1+2)

• Inoculation of ER2738 in 2x 2L conical flask with 1L LB+1ml Tet, over night in 37°C room
• send to sequencing:

•  concentrated pET39b+ ssDNA via sodiumacetat and ethanol precipitation
•  electrical trafo of the 449 ago phagmid bibliothek into XL1
•  prepaired immutubes with streptavidin for ago phages test panning tomorrow

10.10.09 Julia, Caro, Laura, Christoph

• Measured OD600 of over night ER2738 culture abs.1)0.218 (1:10);2)0.23 (1:10), diluted to OD600 abs.0,1 in 1000ml LB+Tet
• Measured OD600 of Christophs culture:abs. 0.37 (1:10), diluted to abs.0.07 in 60ml DYT+Tet+CM
• Growed Er2738 up to OD600 abs.0.6 8x 250ml in 1L Erlenmeyer flasks and infected with 15µl M13 phage, shake for 4 hours at 37°C
• Decanted to 16x50ml falcon tubes, centrifuged for 20min at 5000rpm and 4°C, decanted supernatant into new 16x50ml falcons with each 7ml PEG/NaCl
• Precipitaion over night in 4°C room
• plasmidpreparation of - pBADQuick clones 1-6

• digest for recloning of pMA constructs: pMAShortlinkerFoka clone 1, prep from 03.10.09, pMAMiddlelinkerFoka clone 2, prep from 03.10.09, pMALonglinkerFoka clone 1, prep from 10.09.09, pExDsbAHisDigSplitFoka, clone 1 from 02.10.09, pExDsbAStrepDigSplitFoka, clone 1 from 02.10.09: 10µl

• PCR assembly with pMADsbA, different approaches (Taq Polymerase or Phusion Polymerase, differnt Tm) and combination PCR with pMADsba and pMAFos together
• analytical gel of PCRs -> didn't work
• preparative gels of digests -> see picture -> no inserts with pExDSba_Foka constructs because they have no NgoMIV site any more
• ligation of -pMAStrepDigShortLiFoka

• transformation of ligations in XBL on LB/Amp/1%glucose plates
• cotransformation of
• testdigest of the pET39b+ ssDNA. looks like a success... see 4pk in picture

gele of the AGO pETb+ ssDNA cleavage assay. Lanes: Marker ,ssDNA,1,3,2,4,1pk,3pk,2pk,4pk,ssDNA

• cotransformation of pExHisFluASplitFoki and pJSStrepDigSplitFoka in XBL on LB/Amp/CM/1%glucose plates
• inoculation of pExDsbAStrepDigSplitFoka and pExDsbAHisDigSplitFoka, glystock from 02.10.09

11.10.09, Timo, Hannes, Max, Anika

•  Digestion of 

• gel extraction of - pMA

•  Testdigestion of 

•  1% Agarosegel of constructs above
•  Phage breeding day 2
•  Starter culture of pEx_DsbA_StrepDigSplitFoka (Bl21de3)
•  plasmid prep of pEx_DsbA_StrepDigSplitFoka
•  plasmid prep of pEx_HisDigSplitFoka
•  M13 ssDNA produced with bacterial of Julia and tried another variance with 100ml+tet(1:1000)+ER2738 grow to OD 0,2 then infected withM13phage particles and let it grow for 2h. After this followed the qiagen M13 protocol for M13Dna

12.10.09, Laura, Caro, Christoph, Anika, Hannes, Julia, Timo, Gerrit

•  protein expression of pEx_DsbA_StrepDigSplitFoka (periplasm) in BL21de3 at 22°C.
•  made 5 litres of DYT
• digest of pMASplitFoka clone 1 and 2 from 04.10.09 DNA:10µl Enzymes: 1µl of NgoMIVI and 1.5µl PstI

•  Plasmidprep. of:

• Made new BB-AGO PCR using digested AGO Gene without the vector-> still no expected bands to be seen...
• Made new PCR to generate more pET39b+ ssDNA because yesterdays had insufficient concentration for the AGO cleavage assay
• Made digest of errorprone PCR product of the AGO-G3P constructs (from 02.10.; one with, one without Amber) via NheI and SfiI to gain new Phagmid library
• Send to sequencing:[[Protocols#DNA_Sequencing]]

• Prepared ELISA[[Protocols#ELISA]] and two immuno tubes for Panning Simulation for tomorrow
• made chemical competent cells of cotransformed XBlue with pJS419HisDigSplitFoka, pExHisFluASplitFoki ->will prepare electrocompetent cells tomorrow
• inoculation of cotransformed XBlue with pJS419HisDigSplitFoka, pExHisFluASplitFoki in LB+Amp+Tet+CM and pMAFos in LB+Amp
• PCR of pMADsba repeated with different (higher) template dilutions ->analytical gel (see picture)

• poured IPTG/XGal plates for in vivo plaque assay test run
• transformation of:

• cultivation of pma strep dig split foka

13.10.09, Laura, Caro, Christoph,Manu, Hannes, Julia

• Miniprep -pMA-fos 1

• testdigest of Plasmidprep from today 15 µl for 6µl loading dye -buffer 2 1µl

• made electrocompetent XL1 Blue cotransformed cells (pExHisFluASplitFoki and pJS419HisDigSplitFoka) ->stored in -80°C
• culture of ER2738 in LB Tet for cotransformation in afternoon
• test in vivo assay - electroporation with new electrocompetent cells and M13 ssDNA at 1.7kV

• inoculation of -pMASplitFoka clone 1 from prep 04.10.09

• protein purification of DsbA_StrepDigSplitFoka with Strep column
• digestion, 1% agarose gel and gel extraction of - pMA-LongLinker-FokA (10.09., clone 1) with EcoRI + SpeI

• ligations: Vector: 3 µl pMA (01.10., c=500ng/µl) with EcoRI + SpeI

• did transformations of the ligations in RV308, plated on LB-Amp + 1% Gluc
• ELISA -Blocking

• Made new "AGO BB PCR" this time just using the ad-on-tail-primer for the bb pre- and suffix, so that hte EcoRI site should remain. Still the same not fitting bands were obtained-> the problem might be caused by multimerising of the primers, even so we were not able to predict any of tose using vectir nti or compairable programms...
• AGO assay using different targets-> were not able to reproduce the presumed cutting event from Saturdays assay probably due to the lower DNA concentration
• Made ELISA with strep-biotinylated target oligo coated surface-> no binding of AGO bearing phages was detected using anti-M13 HRP and ABTS
• Made test panning just like the ELISA above and eludet with TBS-MnCl2 (5mM), then TBST-MnCl2 (5mM) and then with DNase I
• Ligated product of the errore-prone PCR of the AGO-G3P construct into an phagmidvektor with the Tor-A signalling sequence

14.10.09, Laura, Caro, Christoph, Hannes, Julia, Anika

* Made LB-medium, 0,7% LB-Agar, 50% Glucose * Test expression (15ml) of pBAD_CAT and pBAD_FokA in BL21de3

OD measurements	1 hour before	induction	45min	1.5h	2h 45min
	induction
1. clone1 pBAD_CAT + 0.6mM IPTG + 0.2% Arabinose	0.1	0.74	1.19	1.25	2.34
2. clone1 pBAD_CAT + 0.6mM IPTG + 0.002% Arabinose	0.1	0.67	1.08	0.94	2.20
3. clone 1 pBAD_CAT control	0.07	0.72	1.08	1.24	2.23
4. clone2 pBAD_CAT + 0.6mM IPTG + 0.2% Arabinose	0.07	0.63	0.96	1.11	2.00
5. clone2 pBAD_CAT + 0.6mM IPTG + 0.002% Arabinose	0.07	0.66	1.06	1.25	2.21
6. clone2 pBAD_CAT control	0.08	0.72	1.11	1.33	2.25
7. clone1 pBAD_Foka + 0.6mM IPTG + 0.2% Arabinose	0.07	0.67	1.13	1.26	2.07
8. clone1 pBAD_Foka + 0.6mM IPTG + 0.002% Arabinose	0.09	0.67	1.21	1.28	2.22
9. clone1 pBAD_Foka control	0.09	0.7	1.11	1.38	2.31
10. clone2 pBAD_Foka + 0.6mM IPTG + 0.2% Arabinose	0.07	0.6	0.93	1.16	1.87
11. clone2 pBAD_Foka + 0.6mM IPTG + 0.002% Arabinose	0.07	0.62	0.94	1.18	1.93
12. clone2 pBAD_Foka control	0.01	0.64	1.04	1.24	2.27

--> no differences between induced and control cells
[[Image:Freiburg09_141009_pg_testexpression1015.jpg|none|thumb|SDS gel of testexpression with pBAD_CAT in BL21de3; lanes: NEB prestained protein marker broad range, clone1 pBAD_CAT+0.6mM IPTG+0.2% Arabinose, clone1 pBAD_CAT control, clone1 pBAD_CAT+0.6mM IPTG+0.002% Arabinose, empty lane, clone2 pBAD_CAT+0.6mM IPTG+0.2% Arabinose, clone2 pBAD_CAT control, clone2 pBAD_CAT+0.6mM IPTG+0.002% Arabinose, empty lane, NEB prestained protein marker borad range|400x400px]] [[Image:Freiburg09_141009_pg_testexpression2016.jpg|none|thumb|SDS gel of testexpression with pBAD_Foka in BL21de3; lanes: NEB prestained protein marker broad range, clone1 pBAD_Foka+0.6mM IPTG+0.2% Arabinose, clone1 pBAD_Foka control, clone1 pBAD_Foka+0.6mM IPTG+0.002% Arabinose, empty lane, clone2 pBAD_Foka+0.6mM IPTG+0.2% Arabinose, clone2 pBAD_Foka control, clone2 pBAD_Foka+0.6mM IPTG+0.002% Arabinose|400x400px]]


* SDS gel of DsbA_StrepDigSplitFoka purification from 13.10.09 [[Image:Freiburg09_141009_pg_pEx_DsbA_StrepDigSplitFoka_Elution017.jpg|none|thumb|SDS gel of protein expression and purification with Streptag column of pEx_DsbA_StrepDigSplitFoka; lanes: NEB prestained protein marker broad range, elution fraction 1, elution fraction 2, elution fraction 3, elution fraction 4, elution fraction 5, elution fraction 6, elution fraction 7, elution fraction 8, elution fraction 9|400x400px]] [[Image:Freiburg09_141009_pg_pEx_DsbA_StrepDigSplitFoka2018.jpg|none|thumb|SDS gel of protein expression and purification with Streptag column of pEx_DsbA_StrepDigSplitFoka; lanes: NEB prestained protein marker broad range, flow through fraction 1, flow through fraction 2, flow through 3, flow through fraction 4, washing fraction 1, washing fraction 2, washing fraction 3, unpurified periplasm extract (frozen at -80°C)|400x400px]] * Plasmidpreparation 1 = pEX-fos-split-fokA-K1
2 = pEX-fos-split-fokA-K2
3 = pMA-DsbA-strep-dig-split-fokA-K1
4 = pMA-DsbA-strep-dig-split-fokA-K2
5 = pMA-DsbA-his-dig-split-fokA-K1
6 = pMA-DsbA-his-dig-split-fokA-K2
7 = pMA-fos-split-fokA-K1
8 = pMA-fos-split-fokA-K2
9 = pBAD-fokA-K1
10= pBAD-fokA-K2
11 = pBAD-CAT-K1
12= pBAD-CAT-K2
13= pMA-shortlinker-fokA
14= pMA-longlinkerFoka
15= pMA-strep-dig
16= pMA-middlelinker-fokA
18=pMA-split-fokA
->sequencing of clones 1 and 2 of pBAD_Foka
*Pellets of: 19=pMA-fokA
20=pEX-DsbA-strep-dig-split-fokA
21=pEX-strep-fluA(3.10)
*digest1: - pBAD-iGEM(Midi 11.10)
- pMA-DsbA-his-dig-split-fokA (Plasmidprep 14.10 Nr5)
- pMA-DsbA-strep-dig-split-fokA (Plasmidprep 14.10 Nr4)

-10µlDNA
-3µl buffer3
-0.5µlBSA(100fold)
-14.5µl water -1µl EcoRI
-1µl PstI
- loaded on 1% agagel *digest2: - pMA-fos-split-fokA (Plasmidprep 14.10 Nr7)
- pEX-fos-split-fokA-K1

-10µlDNA
-3µl buffer4
-0.5µlBSA(100fold)
-14.5µl water -1µl xbaI
-1µl NgoNIV
loaded on 1% agagel *digest3: -pMA-DsbA-clone2 (5.10)

-10µlDNA
-3µl buffer4
-0.5µlBSA(100fold)
-14µl water -1µl xbaI
-1.5µl AgeI
loaded on 3% agagel *Gelextraction -pBAD iGEM
-DsbA-his-dig-split-foka
-DsbA-strep-dig-split-foka
-pMA-fos-dig-split-foka
-pEX-fos-split-foka
--> Picture
*Ligation -pBAD-DsbA-his-dig-split-fokA
-pBAD-DsbA-strep-dig-split-fokA
- pMA-fos-dig-split-fokA-DsbA
-pEX-fos-split-fokA-DsbA
-10µl Qick ligase buffer
-1 µl Quick ligase
-6µl Insert
-3µl Vector
* Transformation of the ligation above in X1Blue competent cells
-100µl and rest of each transformation were plated on Amp/Gluc plates *made top agar (0.7% agar) *nothing grew on IPTG/Amp plates for test in vivo assay -> made control assays with
- electroporating cotransformed XL1Blue with ssM13DNA and plating them directly to IPTG/Amp cells (no phage precipitation and infection of ER2738) to test electrocompetence
- infection of ER2738 with phages from lab phage stock and plating them to IPTG/Amp cells to test plates
->put plates in 37° room overnight *inoclutaion of ER2738 from lab stock and our stock in LB+Tet ->put them on shaker at 26°C * XL1 was el. transformed with the ligated AGO-Phagmid library (errore-prone PCR of the AGO-G3P construct into an phagmidvektor with the Tor-A signalling sequence with and without amberstop from yesterday) and plated everything (4 ml)on two big tubs. * new PCR for ssDNA from the digested pET39b+ vector to get new target for an additional AGO assay *prepaired ELISA to repeat yesterdays AGO-Phage-ELISA tomorrow (because yesterdays results were pretty contradictory -> we probably made a mistake somewhere)

15.10.09, Laura, Caro, Julia, Manu, Timo, Sarah, Hannes, Max, Anika, Christoph

*ELISA:
-Blocking: 350µl/well 1% BSA in TBST-EDTA, incubated at 20°C for 2h gently shaking
-5x washing with 200µl/well TBST-EDTA
-Incubation of phages with oligos for 30min in 55°C waterbath
-Incubation of 75µl/well phages for 1h at 20°C gently shaking
-5x washing with 200µl/well TBST-EDTA
-Antibody: add 75µl/well of anti- M13 antibody (1:1000)
-5x washing with 200µl/well TBST-EDTA
-Substrate: add 75µl/well 1mg/ml ABTS; measure absorbance immediately and after 3 and 5 min; 405nm
*made chemical competent XL1 Blue *test digests of plasmid preparations from 14.10.09 *seqencing of - laura1:pExFosSplitFoka clone 2
- laura2:pMAFosSplitFoka clone 2
- laura3:pMADsbAHisDigSplitFoka clone 2
- laura4: pMADsbAStrepDigSplitFoka clone 1
*inoculation of - ER2738 from our stock
- XL1 blue from our stock
-> in 50 ml LB+Tetracyclin each, put in 37°C *inoculation of -pBAD_Foka clone 1 and 2 from 14.10.09
-pMAFoka clone 1from 04.10.09
-pDsbA clone 2 from 05.10.09
*for in vivo assay - measured pd.7.phage display library concentration new
- mixed 1:10^4, 2*10^6, 2*10^7, 2*10^8 dilutons with ER2738 (OD600 of 0.46) and XL1 blue (OD600 of 0.54) each
- let mixtures stand for 10min (infection time)
- mixed cells with 3ml topagar each and plated them on 3ml IPTG/XGal plates
- put it in 37°C roomin the dark
-pExStrepFluASplitFoki from 08.10.09
-> in 5 ml LB+Ampicillin each, put in 37°C * new ssDNA production via pET39b+ digestion product one-primer-pcr * elusions from the test panning was given to xl1-> infection-> plated in tet cm -> cells infected with the phagmid should grow->tomorrow picking clones and determination of the phagmidinsert *new AGO BB pcr usind dmso, the gene cut out of the vektor and only the ad-on-tail-primers *starter cultures of AGO Aa and AGO Tt in BL21de3 in DYT+Kan *starter cultures of pEx-His-Fos-Split-Foka in RV308 in DYT+Amp+1% Glucose

16.10.09, Laura, Caro, Julia, Manu, Timo, Sarah,Isabelle, Hannes, Max, Anika, Christoph

*Made 10 L DYT
*Inoculation of elutions from panning: E1#2 (MnCl2+TBS)n°1-20, E2#1 (MnCl2+TBST)n°1-20, E3#1 (DNAse)n°1-20

17.10.09, Laura, Julia, Manu, Hannes, Gerrit, Christoph

*Expression of Ago Tt in Bl21de3 OD measurement

	1 hour before
	induction	time of induction	1.5 hours	4 hours	6.5 hours	8 hours
flask 1	0.29	0.53	0.86	1.18	1.32	1.51
flask 2	0.31	0.57	0.91	1.21	1.38	1.49
flask 3	0.32	0.51	0.9	1.31	1.32	1.53
flask 4	0.28	0.49	0.88	1.15	1.33	1.57
flask 5	0.27	0.51	0.89	1.26	1.32	1.48
flask 6	0.32	0.51	0.83	1.24	1.5	1.61

*Dilution from XL1 from OD600 2,8 to OD600 0.07 *Minipreps from : E1#2 (MnCl2+TBS)n°1-20, E2#1 (MnCl2+TBST)n°1-20, E3#1 (DNAse)n°1-20 wich are phagemids we cultured from elutions of panning from 13.10.09
*IPTG/X-Gal plates with infected ER2738 was blue, with the infected XL1 blue no blue plaques could be seen -> so infection by phages was positive and lots of blue plaques were built up [[Image:Freiburg09_17-10-09_Phagenplatten_XGal.JPG|none|thumb||400x400px]] *made new in vivo test assay with cotransformated, electrocompetent XL1 blue - transformed M13 ss DNA from 17.08.09 into cells by electroporation at 1.7kV
- 1.5h incubation time on 37°C shaker
- 80, 10 and 5µl of electroporated cells were mixed with 180µl XL1 blue and ER2738 with OD 600 of about 0.5
- incubation time of 10 min at room temperature
- mixing cells with 3ml topagar, vortexing shortly and plating on IPTG/X-Gal plates
- put in 37°C room overnight
*control of electrocompetence of XL1 blue by electroporation of pET28a with Kan resistance into XL1 blue and RV respecitvely -> comparison in the end or if colonies will show up at all will show if our cotransformated cells are competent at all *inoculation of - pMAFoka clone 1 from 04.10.09
- pMADsbA clone 2 from 05.10.09
- pBADFoka clone 1 from 15.10.09
- pExStrepFluALongliFoki clone 1 from 08.10.09
*miniprep of all clones picked from the elutions of the test panning to determine the efficiency of the selection *new ssDNA via Pet39b+ one primer pcr *new infection of XL1 cells with the elution from the test panning-> this time plated on lb agar with tet cm and GLUC (to suppress the expression of the phagmid insert)

18.10.09 Manu

*Expression of Ago Aa at 24°C *Expression of GST-FosW at 30°C

19.10.09, Laura, Caro, Julia, Manu, Timo, Sarah, Isabel, Hannes, Max, Anika, Christoph

* analysis of the geles of the digestion of the clones picked from the elution plates revealed not a single clone containing the ago insert-> possibly the lack of gluc on the plate led to a selection against this insert-> new klones picked from the plate with gluc *new ago cleavage assay reveald last saturdays promising results as false. it seems like it was just two conformations of one ssDNA molecule * starter cultures of pEx-His-Fos-Split-Fok_a * protein purification of GST-FosW with Ni-NTA column - French pressed, centrifuged 15min at 20000rpm, sonicated and filtered
- column was washed with 25mM imidazole and eluted with 250mM imidazole * in vivo assay retried with shorter incubation time

20.10.09 Manu, Laura, Anika, Julia, Timo, Sarah, Isabel, Max, Gerrit, Christoph, Caro, Hannes

* new round of expression with pEx-His-Fos-Split-Fok_a in BL21de3at 24°C

OD measurement	time of	2 hours	3 hours	3 hours	4 hours
	induction			45 min	15 min
flask 1	1.3	2.63	3.47	4.29	3.99
flask 2	1.19	2.87	3.87	4.51	4.62
flask 3	1.5	3.22	4.45	5.03	5.41
flask 4	1.03	2.9	4.21	5.11	5.88
flask 5	0.81	2.81	3.76	4.29	4.76
flask 6	1.52	2.72	3.71	3.95	5.17