Team:Goettingen/NoteBook w13

Sequencing results: G2L, Promoter 3rev in part 6.2

-          for all plasmids, the sequence stops at the same region as for the seqlab sequences

-          DMSO and denaturing didn’t help…

→ sequencing of Promoter3rev in pSB1C3 with VF2 and VR (from behind insert) and sequencing of Promoter3rev in part 6.2 with reverse qRT-PCR GFP primer…

Transformation of ligations from 27.8.13 (DarR reporter system)

-          comp. cell strain: XL1-Blue

-          for neg. control, 20 μl sterile dH2O were added to the cells

-          whole ligation reactions were transformed

-          removal of 500 μl supernatant after centrifugation

-          plating on LBCm plates

-          incubation ON at 37 °C (started at ca. 11:20 a.m)

Harvesting of cells for qRT-PCR analysis and plate reader assay (promoter clones)

-          measuring of OD600nm of ON cultures in 1:20 dilution of LB and calculation of actual OD600nm→ OD600nm in ON cultures varied from 3.5 to 3.84 → inoculation of synchronization pre-culture: 4 ml LBAmp + 50 μl ON culture, incubation at 37 °C at ca. 200 rpm for > 2,5 h (in between, the OD600nm was measured and the cells kept on ice → don’t keep cells on ice!!! keep them on bench!)

-          measuring of OD600nm in 1:10 dilution of LB and calculation of actual OD600nm 

-          preparation of plate reade titer plate (170 µl culture) and 100 ml Erlenmeyer flask w/o baffles (15 ml culture), both cultures: OD600nm = 0.05

a) OD600nm ON culture

b) measurement of synchronization culture in between

c) measurement of synchronization culture before main culture and titer plate preparation

The volumes indicated in the table were added to 160 µl or 15 ml LBAmp, respectively

*calculation: Volume (of new culture) * OD600nm (of new culture)/OD600nm (old culture) = Volume (old culture, needed for preparation of new culture)

qRT-PCR: Harvesting of Cells

-          measuring of OD600nm in 1:10 dilution of LB and calculation of actual OD600nm→ inoculation of main cultures: 15 ml LBAmp, OD600nm =0.05 (approx.)

-          incubation at 37 °C, 200 rpm

-          growth curve → measuring of OD600nm in 1:10 dilution of LB and calculation of actual OD600nm over time, plot of Log2 (OD600nm) against time (in h)

-          harvesting of 2 ml culture at an OD600nm = ca. 0.6 (log phase) by centrifuging for 3 min at 14.8 rpm, 4 °C, then supernatant taken off completely and cell pellet frozen in liquid nitrogen, storage at – 80 °C, harvesting of 1 ml culture at an OD600nm = ca. 2 (stationary phase), as described before

-          after harvesting 1 ml, the cells were incubated ON (in total ca. 20 h)

Plate Reader assay

-          measuring of OD600nm in 1:10 dilution of LB and calculation of actual OD600nm→inoculation of plate reader wells: 170 μl LBAmp, OD600nm = 0.05 (approx)

-          incubation at 37 °C, strong shaking

-          measuring of OD600nm against LBAmp as blank

-          measuring of RFP fluorescence (excitation: 555 nm; emission: 583 nm)

-          run over day and ON

-          pipetting scheme (I made a mistake and pipetted a higher volume of P4 C1 culture in row 6, correct volume of P4 C1 culture was added in row 7)

RiboA Cells inocculated yesterday did not grow except for C5.8 from yesterday´s overday-culture picked from the plate from the 21.8.

Discussion: RiboA C5.8 will be our Antibiotic Detector!

“Drop”-experiment with the DAC and empty vector control plates made by Jan yesterday

4 plates:

1x Gp1013 without IPTG

1x Gp1013 with IPTG

1x p172 without IPTG

1x p172 with IPTG

On each plates 3 drops of RiboA:

1x undiluted overnight culture

1x 1:10 diluted overnight culture

1x undiluted overnight culture smeared over one half of the plate

Plates incubated at 37°C overnight

Preparation of RiboA C5.8 cells for plates as already made by Jan yesterday

Retrafo of RiboA C5.8 from Mini-prep from (DATE?)

Inocculation of the DAC cells, the empty vector control for the second Drop”-experiment and the CFP w/o insert religation control as a control for our detector

Preparation of 2x 500 ml LBAmp plates (stored in cold room, “Görke” shelf)

Test gel for DarRrev-Termrev C3 vector after 2nd digest (with PstI)

-          1 % agarose-1x TAE gel

-          loading of 3 μl 2 log ladder

-          loading of 3 μl RD reaction + 1 μl dH2O + 1 μl 5xLD

-          loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

-          rut at 100 V

-          EtBr staining + destaining in water

-          UV detection 

loading: Marker/uncut DarRrev-Termrev C3 plasmid/ RD DarRrev-Termrev C3 vector

digest still partial (expected band at ca. 3kb is observed, but also a weak band at ca. 6 – 8 kb of uncut plasmid) → dephosphorylation of vector with AP to avoid self-ligation

Preparation of LB media

-          1x 300 ml broth LB w/o antibiotics

-          1x 500 ml LBCm plates

Dephosphorylation of DarRrev-Termrev C3 vector cut with SpeI and PstI

ca. 37 μl digest reaction

+ 2 μl AP

+ 5 μl 10x AP buffer

+ 6 μl dH2O

incubation for 1.5 h at 37 °C (PstI forms 3’ overhang, but dephosphorylation often incomplete → incubation for more than 1 h)

Preparation of CryoStocks of clones inoculated yesterday

DMSO cryostocks prepared as described before (13.08.2013)

for all clones inoculated on 26.8.13:

-          part 1 C2

-          part 2 C3

-          part 3 C2 and C3

-          part 4 C2 and C3

-          DarRrev-Termrev in pSB1C3 C3

-          part 6.4 A C3

-          part 6.4 B C2 and C5

-          Promoter1rev in pSB1C3 C4

-          Promoter3rev in pSB1C3 C5

Plasmid Mini Preparation 

-          harvesting of all cultures inoculated yesterday

-          but MiniPrep only for the following clones:

DarRrev-Termrev in pSB1C3 C3

part 6.4 A C3

part 6.4 B C2 and C5

Promoter1rev in pSB1C3 C4

Promoter3rev in pSB1C3 C5

-          all other clones were harvested and the cell pellets stored in to-do-box (it could be that we won’t need the plasmids…):

part 1 C2

part 2 C3

part 3 C2 and C3

part 4 C2 and C3

-          elution 1x with 30 μl HPLC water (pre-warmed), incubation at 50 °C for 2 min

-          NanoDrop concentration measurement:

Test gel for plasmids after Plasmid Mini Preparation and part 6.4 A/B vectors

-          1 % agarose-1x TAE gel

-          loading of 3 μl 2 log ladder

-          loading of 3 μl RD reaction + 1 μl dH2O + 1 μl 5xLD

-          loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

-          rut at 100 V

-          EtBr staining + destaining in water

-          UV detection 

loading: Marker/DarRrev-Termrev C3 plasmid/ part 6.4 A C3 uncut plasmid (from today’s prep)/ part 6.4 A C3 vector (E + X)/part 6.4 B C2 uncut plasmid (from today’s prep)/ part 6.4 B C2 vector (E + X)/ part 6.4 B C5 plasmid/Marker

→ for vector digests, the expected bands were obtained (ca. 3 kb), but the digestes are still slightly incomplete (weak 2 kb band) → dephosphorylation with AP

→ plasmids purified today look normal

Dephosphorylation of part 6.4 A and part 6.4 B vector with AP

ca. 37 μl digest reaction

+ 2 μl AP

+ 5 μl 10x AP buffer

+ 6 μl dH2O

incubation for 1 h at 37 °C (EcoRI and XbaIform5’ overhang, but dephosphorylation often incomplete → incubation for more than 15 min)

Test RD of Promoter1rev and Promoter3rev in pSB1C3 

2 μl plasmid (from today’s purification, ca. 200 – 300 ng plasmid)

1 μl EcoRI FD

1 μl PstI FD

1 μl 10x FD Green buffer

5 μl dH2O

in total: 10 μl

-          both reactions were pipetted individually

-          incubation for 1 h at 37 °C

Gel run: Test RD of Promoter1rev and Promoter3rev in pSB1C3

-          1 % agarose-1x TAE gel

-          loading of 3 μl 2 log ladder

-          loading of 5μl RD reaction

-          loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

-          rut at 100 V

-          EtBr staining + destaining in water

-          UV detection

Loading: marker/uncut Promoter1rev C4 plasmid/ RD Promoter1rev C4 plasmid/ uncut Promoter3rev C5 plasmid/ RD Promoter3rev C5 plasmid

→ both digests are only partial

→ but the expected bands were obtained: 112 bp Promrev + 2x20 bp prefix/suffix = 152 bp and ca. 2 kb of pSB1C3 backbone

→ sequencing of both plasmids

PCR clean-up of vectors (part 6.4 A and part 6.4 B vector; DarRrev-Termrev C3 vector) after AP treatment

-          with Qiagen PCR purification kit

-          addition of 500 μl PB buffer

-          elution with 30 μl HPLC water, incubating for 2 min at 50 °C, centrifugation by mistake for 2 min at 13 000 rpm

-          NanoDrop concentration measurement:

Plates Prepared for the Drop experiments

4 plates:

1x Gp1013 without IPTG

1x Gp1013 with IPTG

1x p172 without IPTG

1x p172 with IPTG 

As follows:

25ml sterile water were filled intotwo 50ml falcons. Also two of the 300µl Glycerin stocks (Gp1013 and p172) prepared earlier were added to the water in the dedicated falcons. The falcons were inverted several times. 2x LB medium was prepared by Katrin Gunka and melted in the microwave. It was added to the 25ml water rather hot to the total volume of 50ml. The falcons were inverted a few times and then one 25ml plate was poured from each falcon. Then, very fast, where needed, 25µl IPTG 1M (end concentration 1mM) were added.

Inoculation of Promoter clones for Plate Reader Assay and RT-PCR analysis

-          inoculation of

part 1 C1

part 2 C1

part 2 C2

part 3 C1

part 4 C1

part 8 C1 (as empty vector control, AmpR, but no RFP, only RBS)

-          in LBAmp from cryostocks:(scratch some frozen E.coli cells from culture tube with a yellow pipet tip and transfer them directly in LBAmp)

-          incubation ON at 37 °C at 200 – 210 rpm

Concentration of dialysed protein samples

-       Pool all desalted samples from dialysis together (20ml)

-       Prepare vivaspin column: apply 10ml H₂O and centrifuge for 10min at 4000g at 6°C; apply 10ml of 1x dialysis buffer and centrifuge until almost all 10ml have run through

-       Apply 10ml of sample from dialysis (with 2,3 mg/ml) to vivaspin column

-       Centrifuge column at 4000xg at 6°C until protein concentration was 10mg/ml

Determination of final protein concentration via Bradford test

-       Add 1ml of Bradford solution (1:5 dilution = 200ml Bradford + 800ml H₂O) to 10µl of 1:20 diluted protein solution

-       Incubation for 5min at RT

-       Measure extinction at 595nm

-       Calculation: c= OD₅₉₅ / Vx0,0536

SDS Page of elution samples

-       Mix 15µl of samples  with 5µl Pab and 5µl buffer W

-       Incubation for 10min at 93°C

-       Mix each sample with 4µl protein marker

-       Load whole volume on 15% SDS-gel

-       Run for 5min at 90V and 1h at 120V

M | E1 | E2

Sequencing results from 23.8.13

9 – DarRrev-Termrev C2 + VF2→ DarRrev and Terminatorrev are inserted in the plasmid in the desired orientation without mutations

10 – DarRrev-Termrev C2 + VR→ DarRrev and Terminatorrev are inserted in the plasmid in the desired orientation without mutations

11 – DarRrev-Termrev C3 + VF2→ DarRrev and Terminatorrev are inserted in the plasmid in the desired orientation without mutations

12  - DarRrev-Termrev C3 + VR→ DarRrev and Terminatorrev are inserted in the plasmid in the desired orientation without mutations

13 – part6.4 B C2 + VF2→ RBSrev is inserted in the desired orientation, but sequencing stopped again in reverse promoter 3

14 – part 6.4 B C5 + VF2→ RBSrev is inserted in the desired orientation, but sequencing stopped again in reverse promoter 3

15 – part 6.4 A C2 + VF2 → RBSrev is not inserted

16 – part 6.4 A C3 + VF2→ RBSrev is inserted in the desired orientation, but a part of pSB1C3 is missing at the EcoRI restriction site (but EcoRI restriction site is present) and a G of NotI restriction site in prefix is missing…

17 - part 6.4 A C5 + VF2→ RBSrev is not inserted

18 – part 6.4 A C8 + VF2→ RBSrev is not inserted 

Plan:  sequencing of the other 4 part 6.4 A clones and cutting out of part 6.4 A C3 insert with XbaI and PstI (one would get rid of missing/erroneous regions) and integration in vector DarRrev-Termrev plasmid C3; same for part 6.4 B C2

Plates for C1 – C3 of parts 1 – 4

-          today, the part 2 clones C2 and C3 are slightly pink, while C1 is white as C1 and C2 of part 1 (C3 of part 1 never existed…)

-          clones of part 3 and part 4 are very pink

-          pictures were taken:

Restriction digests

a) DarRrev-Termrev in pSB1C3; C3 with EcoRI and SpeI 

3 μl SpeI FD

3 μl EcoRI FD

4 μl FD buffer 10x

7 μl plasmid (229.7 ng/μl, purified on 23.8.13 → ca. 1500 ng)

23 μl dH2O

in total: 40 μl

b) part 6.4 A C3 and part 6.4 B C2 with EcoRI

4μl EcoRI FD

4 μl FD buffer 10x

15μl plasmid (6.4 A: 107.5 ng/μl, purified on 23.8.13 → ca. 1500 ng; 6.4 B: 114.6 ng/μl, purified on 23.8.13 → ca. 1500 ng)

17μl dH2O

in total: 40 μl

Cloning strategy changed -  suddenly new idea occurred to Katrin and me: sequencing of the other 4 part 6.4 A clones and cutting out of part 6.4 A C3 insert with XbaI and PstI and integration in vector DarRrev-Termrev plasmid C3; same for part 6.4 B C2; but to avoid a waste of material, the above mentioned reactions were incubated as well and stored at – 20°C in DarR box (in addition, they can serve as a control for part 6.4A, to see, if EcoRI site is really there!)

c) DarRrev-Termrev in pSB1C3; C3 with SpeI

4μl SpeI FD

4 μl FD buffer 10x

7 μl plasmid (229.7 ng/μl, purified on 23.8.13 → ca. 1500 ng)

25μl dH2O

in total: 40 μl

d) part 6.4 A C3 and part 6.4 B C2 with XbaI and PstI

3μl PstI FD

3 μl XbaI FD

4 μl FD buffer 10x

15μl plasmid (6.4 A: 107.5 ng/μl, purified on 23.8.13 → ca. 1500 ng; 6.4 B: 114.6 ng/μl, purified on 23.8.13 → ca. 1500 ng)

15μl dH2O

in total: 40 μl

→ all reactions incubated for 1.5 h at 37 °C (incubation time decreased since missing part of vector in plasmid part 6.4 A C3 might result from EcoRI FD overdigest within 2 h incubation time…)

Test Gel run for Restriction digests (see above)

-          1 % agarose-1x TAE gel

-          loading of 3 μl 2 log ladder

-          loading of 3 μl RD reaction + 1 μl dH2O + 1 μl 5xLD

-          loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

-          rut at 100 V

-          EtBr staining + destaining in water

-          UV detection

Gel:

Loading: Marker/ part 6.4 A C3 uncut/ part 6.4 A C3 RD EcoRI/ part 6.4 A C3 RD XbaI + PstI/ part 6.4 B C2 uncut/ part 6.4 B C2 RD EcoRI/ part 6.4 B C2 RD XbaI + PstI/DarRrevTermrev C3 uncut/ DarRrevTermrev C3 RD EcoRI + SpeI/ DarRrevTermrev C3 RD SpeI/Marker

→ both digests of part 6.4 A seemed to be complete (expected bands for XbaI/PstI double digest were obtained, i.e. ca. 1 kb and ca. 2 kb band; expected band for EcoRI single digest, i. e. 3 kb band, was also obtained)

→ single digest of part 6.4 B seemed to be slightly incomplete, while double digest appeared to be complete (expected bands for XbaI/PstI double digest were obtained, i.e. ca. 1 kb and ca. 2 kb band; expected band for EcoRI single digest, i. e. 3 kb band, was also obtained)

→ single digest of DarRrev-Termrev seemed to be slightly incomplete, while double digest appeared to be complete (expected bands for EcoRI/SpeI double digest were obtained, i.e. ca. 0.8 kb and ca. 2 kb band; expected band for SpeI single digest, i. e. ca. 2.8 kb band, was also obtained)

-          purification of inserts (except for DarRrev-Termrev insert, stored unpurified at – 20 °C in To-do-box) by gel ex and purification of vectors by PCR clean-up

Purification of vectors from today by PCR clean-up (DarRrev-Termrev vector/part 6.4 A/B vector) after first round of digest

-          with Qiagen PCR purification kit

-          500 μl PB buffer were added to the samples

-          elution with 30 μl pre-warmed HPLC water, incubation for 2 min at 50 °C

-          further digest

Second round of restriction digest to generate vectors for ligation

a) DarRrev-Termrev vector previously cut with SpeI

30 μl linearized plasmid

2 μl dH2O

4 μl PstI FD

4 μl FD buffer 10x

in total: 40 μl

b) part 6.4 A/B vector previously cut with EcoRI

30 μl linearized plasmid

2 μl dH2O

4 μl XbaI FD

4 μl FD buffer 10x

in total: 40 μl

-          incubation of all three reactions at 37 °C for 1.5 h

-          samples stored at – 20 °C in to-do-box

Purification of part 6.4 A/B inserts by gel extraction

-          1 % agarose-1x TAE gel

-          loading of 3 μl 2 log ladder

-          loading of entire RD reaction (ca. 37 μl supplied with 7μl 5xLD)

-          loading of 1 μl uncut plasmid + 3 μl dH2O + 1 μl 5xLD

-          rut at 85 V

-          brief EtBr staining + brief destaining in water

-          short UV detection, then gel extraction as described on 25.6.13

gel after gel ex:

loading: Marker/ uncut part 6.4 A C3/ - /RD part 6.4 A C3/ RD part 6.4 A C3/ RD part 6.4 A C3/ RD part 6.4 A C3/ RD part 6.4 A C3/-/RD part 6.4 B C2/ RD part 6.4 B C2/ RD part 6.4 B C2/ RD part 6.4 B C2/uncut part 6.4 B C2/ Marker

Inoculation of clones for CryoStocks and MiniPrep for Test RD/Sequencing

inoculation of

-          part 1 C2

-          part 2 C3

-          part 3 C2 and C3

-          part 4 C2 and C3

→ in 4 ml LBAmp

→ all for cryo stocks and MiniPrep

inoculation of

-          DarRrev-Termrev in pSB1C3 C3 → for cryostock and MiniPrep

-          part 6.4 A C3 → for cryostock and MiniPrep

-          part 6.4 B C2 and C5 → for cryostock and MiniPrep

-          Promoter1rev in pSB1C3 C4 → for cryostock, MiniPrep, test RD and sequencing

-          Promoter3rev in pSB1C3 C5 → for cryostock, MiniPrep, test RD and sequencing

→ in 4 ml LBCm 

incubation ON at 37 °C, 200  - 210 rpmpurification: no isopronol used since fragments ca. 1 kb; each sample (part 6.4 A or part 6. 4 B) was distributed onto two colums, DNA from each column was eluted with 30 μl pre-warmed HPLC water (incubation of sample for 2 min at 50 °C. The DNA from both columns of one sample was collected in the same tube.

-          NanoDrop concentration measurement:

-          sample stored in To-do-box

Inocculation of Ribo A C5.6 and C5.8 from cryo stocks in LB medium containing Cm

Inoccultion of DAC (GP1013) and the empty vector (pGP172) from cryo stocks in LB medium containing Amp

both stored at 30°C over night in the shaker

Protein purification

-       resuspend cells from one falcon (pellet from 40ml of expression culture) in 10ml buffer W

-       apply solution from each falcon twice to french press

-       centrifugation for 15min at 8500rpm at 4°C

-       transfer supernatant into ultracentrifugation tubes

-       Ultracentrifugation for 1h at 35000rpm at 4°C

-       Preparation of columns: add 1ml of 50% Strep-Tactin and equilibrate with 10ml buffer W

-       Divide supernatant received from ultracentrifugation into 2 equal volumes and fill it up to 30ml with buffer W

-       Load protein solution on prepared columns

-       Load flow through 1 again onto new column

-       Wash 5 times with each 2,5ml buffer W

-       Elute with 0,5ml (for eluate 1) or 1ml (eluate 2-3) of buffer E

Determination of protein concentration via Bradford test

-       Add 1ml of Bradford solution (1:5 dilution = 200ml Bradford + 800ml H₂O) to 3µl of protein solution

-       Incubation for 5min at RT

-       Measure extinction at 595nm

-       Calculation: c= OD₅₉₅ / Vx0,0536

è Have 16x 1ml of E2

Dialysis of protein samples

-       Fill 3 dialysis bags for dialysis with each 5ml of E2

-       Fill 1 dialysis bag with 1ml E2 and 8x 0,5ml of marked E1

-       Put dialysis bags into each 4500ml H₂O + 500ml 10x dialysis buffer

-       Dialysis o/n with agitation