Team:Goettingen/notebook wetlab
From 2014.igem.org
October 6th, week 22
S. cerevisiae
- Continue the robot Y2H (plate on to SC-LWH + 3mM 3-AT plates and X-α-gal plates)
September 22rd, week 20
S. cerevisiae
September 15th, week 19
S. cerevisiae
- Repeat trafo in Y187
⇒ Trafo worked
September 8th, week 18
S. cerevisiae
- Pick colonies from E.coli plates and isolate plasmids
- Transformation of yeast (Y187)
⇒ Trafo failed
September 1st, week 17
S. cerevisiae
- Spot positive clones on SC-LWH plates with 3mM 3-AT
- Wash cells from plates and isolate plasmids
- Transformation into DH5α
August 25th, week 16
S. cerevisiae
- Y2H in semi solid media with the two genes
August 18th, week 15
C. albicans & C. glabrata
PCR with different plasmid isolation samples for prey amplification (IG1003 and IG0099; 52,5 °C)
Agarose gel (1%):
⇒ Bands in all samples
⇒ Purification and restriction with HpaI and BamHI
- Restriction of of 0,1 µl (14500ng/µl) pGP172 with SmaI and BamHI o/n 16°C
- ligation of cut cleaned PCR prey products with pGP172 (10ng/µl) 4µl of each with 1µl T4 Ligase in 20 µl sample
- Transformation into DH5α
- Again: plasmid isolation and PCR with T7 promotor and terminator primer on plasmids
Agarose gel (1%):
⇒ In lines 4 to 9 seems to be the right fragment; 2 and 3 are wrong
- Transformation into BL21
- Test-expression of strep-tag strains: 4.1A, 4.1B, 5A, 5B, 14.5A, 14.5B, 14.8A, 14.8B
- Preparation of SDS gels
- PCR with Primers IG1003 and IG0099 → product that can be sequenced and cut with enzymes for strep tag plasmid insertion (samples of prey from 3, 13 and 15)
Agarose gel (1%):
⇒ Bands in lines 1, 2, 3, 4, 5 6, 9 and 11 have the right size and are first purified and then send for sequencing with Primer IG1003 (rev)
- Prey samples of 3A.1, 3A.2, 3A.3, 3B.1, 3B.2, 13.3, 15 digested with BamHI and HpaI
⇒ Clear difference between cut and uncut; also cut bands are very weak
- Results of sequencing (3, 13, 15):
⇒ 3A.1, 3A.2, 3A.3 are the same peptide
⇒ 3B.1 and 3B.2: sequences are not useful
⇒ 13.3 and 13.4: same peptide
⇒ 9: one interacting peptide
⇒ 10 + 15: same interacting peptide→?
August 11th, week 14
C. albicans & C. glabrata
- Yeast Plasmid isolation of preys for: 01, 02, 06, 08, 12, 13, 15, 16 (pIGX)
NanoDrop:
- Preparation of fresh competent cells
- E.coli Transformation: supercompetent cells and fresh competent cells are transformed with different plasmids (see table)
- Results of E.coli transformation:
⇒ Plasmid isolation and transformation into Yeast Y187 → but no colonies
August 4th, week 13
C. albicans & C. glabrata
July 28th, week 12
C. albicans & C. glabrata
- Yeast transformation (Y187) from preys of 01, 02, 02C, 03A+B, 06, 07, 13, 15, 16, 16C
⇒ no colonies for 02C, 06 and 16C
- Plasmid multiplication and isolation of prey plasmids from 04, 05, 09, 10, 14
S. cerevisiae
- Yeast transformation (AH109) of pIGB22 and 21
July 21th, week 11
C. albicans & C. glabrata
- Evaluation of Sequencing:
- Bad/ not useful sequences: 9, 10, 14.1, 14.3, 14.4, 14.7, 4.2 (4.2→ new PCR necessary, wrong fragment)
- Peptides from: 4.1, 5, 14.5, 14.6, 14.8 (=14.10), 14.9
July 14th, week 10
C. albicans & C. glabrata
- Confirmed interactions from robot Y2H are used for Plasmid isolation (prey): 4.1; 4.2; 5; 9; 10; 14.1; 14.3; 14.4; 14.5; 14.6; 14.7; 14.8; 14.9 and 14.10 + Isolation of interacting preys of AIG003, 07, 08, 12, 13
- NanoDrop
- additional plasmid purification for sequencing of plasmids 4.1, 4.2, 5, 9, 10, 14.1
- all plasmids are used in a PCR with Primers IG1002 (1170_fwd) and IG1003(1170_rev) (56°C)
Agarose gel (1%):
⇒ PCR products with IG1003 for sequencing (line 3→ fragment bigger than others, but also sequenced)
- Transformation E.coli DH5α with 03, 07, 08, 12, 13
- Plasmid isolation of E.coli 02, 06, 15, 16, 2C, 16C
NanoDrop:
July 7th, week 9
C. albicans & C. glabrata
- Plasmid isolation of prey interactions of 02, 06, 15, 16 and transformation into E.coli (Amp plates)
- Another Y2H is performed with AIG004, 05, 09, 10, 14 and YIG04, 05, 09, 10, 14 to confirm the interactions we saw in the first Y2H (using a robot); testing on SC-LWH plates with 3mM 3-AT and X-α-Gal plates
A. fumigatus & A. nidulans
A. fumigatus
- Plasmid isolation of the genes 02, 06, 15, 16
- NanoDrop:
- 3rd Y2H is performed with the strains AIG003, 07, 08, 12 and 13
June 30th, week 8
C. albicans & C. glabrata
- 1st) Transformation in yeast: some colonies are grown, but not for all → tried transformation again
- Plasmid isolation and saving of prey strains on SC-L plates
- 2nd) Plasmid isolation from yeast for prey plasmids interacting with AIG002, AIG006, AIG015, AIG016 (piGX plasmids)
- Transformation in DH5α → not successful (next week again)
- Third Y2H screening of AIG003 (sim1), 07 (pir3), 08 (utr2), 12(ecm33), 13 (sun1)
A. fumigatus & A. nidulans
A. fumigatus
- Yeast Trafo into Y187 again
- 2nd Y2H: Plasmid isolation for Transformation into E.coli (pIGZ)
- Safe prey interacting partners on SC-L
- Preparation of new AA mixture
June 23rd, week 7
C. albicans & C. glabrata
- Spotting of second Y2H (AIG002 (tos1), 06 (scw4), 15 (als3), 16 (rodA)) (picture)
Figure: Different interacting and matted colonies are spotted onto the plate
- 1st) Yeast colonies from first spotting are solved in water and centrifugated to pellets
- Isolation of plasmids from pellets (see protocol) (pIGX plasmids)
- Transformation in DH5α (70 µl of pooled plasmid DNA plated on Amp-plates)
- Thin layer of E.coli cells are washed from plates with 1,5 L of sterile water; centrifugation (4krpm, 5min), plasmid isolation (pIGY plasmids)
- Transformation into yeast (Y187) cells. Plated onto SC-L plates (YIG strains)
- 2nd) Yeast colonies from second spotting are solved in water and centrifugated to pellets → frozen
A. fumigatus & A. nidulans
A. fumigatus
- Preparation of yeast plasmid prep. Solutions P1, P2, and N3
- 1st Y2H: Yeast colonies are washed from plates; plasmid isolation, Transformation into E.coli; E.coli plasmid isolation and again transformation into yeast (Y187) (YIG strains) (see Candida)
- 2nd Y2H: yeast colonies are washed from plates and centrifuged to pellets → frozen
A. nidulans
- E.coli plasmid isolation of cells with LR product
- PCR on plasmids → no positives
- Repeat LR reactions and transformation
- Again plasmid isolation and PCR
Agarose Gel (1%):
⇒ Right bands in: 1, 2, 4, 5, 6, 7, 8, 9, 16
- Plasmid isolation of all positive clones
S. cerevisiae
- PCR on isolated plasmids
⇒ Nothing on Gel
- LR reaction was repeated for CWP1, CWP2, SED1, MID2
- Transformation of DH5 α
⇒ Worked for all, except CWP1
⇒ Cracking failed
- Colony PCR
- NanoDrop:
June 16th, week 6
C. albicans & C. glabrata
- Second Y2H screening for (tos1), 06 (scw4), 15 (als3), 16 (rodA) (titerplates didn’t work)
- From first Y2H screening (AIG001, 04,05,09,10,14): 10 µl of each growing colony from semi solid medium is transferred into 100 µl of SC-LWH → 3µl spotted onto SC-LWH + 3mM 3-AT plates (picture) (rest: glycerol added and frozen)
Figure: Colonies are growing inside the semi-solid medium
A. fumigatus & A. nidulans
A. fumigatus
- Preparation for Y2H
- Second Y2H (see Candida)
- Spotting of colonies from first Y2H
A. nidulans
- Repeat LR reaction and Transformation in fresh competent DH5α cells
⇒ Trafo worked
- Cracking
⇒ No positives → cracking may have failed
⇒ Try with colony PCR → failed
S. cerevisiae
- Transformation of DH5α with LR product
⇒ Trafo worked for all, except CWP2
- Cracking of the colonies
⇒ No positives
- Isolation of plasmids
June 9th, week 5
C. albicans & C. glabrata
- No growth on auto-activity plates
- Preparation of media for Y2H
- First Y2H screening (semi solid medium) of AIG001 (mp65); AIG004 (ssr1); AIG005 (pir4), AIG009 (crf2full); AIG010 (crf2act); AIG014 (als1);
- Titerplates on SC-LW medium
Titerplates:
A. fumigatus & A. nidulans
A. fumigatus
- LR reaction and transformation of pIGA11 and 13
- Transformation into DH5α
⇒ Trafo worked!
- Transformation into Yeast AH109
- First Y2H of AIG09 and AIG10 (see Candida notebook)
A. nidulans
- Repeat BP reactions and transformation into DH5α
⇒ Trafo worked
- Cracking (see Candida)
- o/n cultures of positive clones
Agarose Gel (1%) of cracking:
- Plasmid isolation
- NanoDrop:
- LR reaction with the 4 plasmids
- Transformation into fresh competent DH5α cells
⇒ Trafo failed
S. cerevisiae
- Cracking for CWP1, CWP2, SED1, MID2
Agarose gel (1%):
⇒ All of the E.coli strains contain the right plasmid (band 4 is negative due to pipetting mistake)
⇒ Sample 2 of each gene was chosen for plasmid isolation
- NanoDrop:
- LR reactions
June 2rd, week 4
C. albicans & C. glabrata
- LR reactions with pDEST and pIGA-plasmids and Transformation into DH5α
- Transformation was successful → Cracking + 4 mL LB+ kan o/n cultures
Agarose gels (1%):
- Samples 1 (mp65), 6 (tos1), 7(sim1), 11(ssr1), 13 (pir4) and 15 (scw4) are used for plasmid isolation.
- Samples 2 (pir3), 4 (utr2), 6 (crf2 full), 9 (crf2 act) and 13 (ecm33) were used to isolate the plasmids.
- The samples from 10-12 (bglCno) were cracked again.
- Samples 9 (als3) and 14 (eglC) are used to isolate the plasmids. The samples for sun1, als1 and rodA are cracked again.
- Cracking of each 3 samples of 11(bglEno), 13(sun1), 14(als1), 16(rodA)
Agarose gels (1%):
- Samples 9 (als1) and 12 (rodA) are used to isolate the plasmids. No results for bglEno and sun1 (A. fumigatus)
- Plasmid isolation
- Nano-Drop results:
- Transformation in Yeast (AH109) → plated on SC-W plates
- Transformation worked → single colonies are streaked on SC-W and SC-HW +3mM 3AT (auto-activity test) plates
A. fumigatus & A. nidulans
A. fumigatus
- LR reaction with pDEST and pIGA- plasmids
- Transformation into DH5α
⇒ Trafo worked
- Validation of positive clones via E.coli cracking (from each plate 3 colonies) (pictures see Candida)
- 4 ml LB with kanamycin inoculated for positive clones
- Plasmid isolation (pIGB plasmids)
- NanoDrop: (pIGB11 and 13 were empty)
- Yeast (AH109) transformation (AIG strains)
⇒ Trafo worked
- Test on autoinduction (His auxotrophy) on SC-HW + 3mM 3-AT
⇒ No autoinduction (grid plate see Candida labbook)
A. nidulans
- Amplification of GOI (size)
⇒ A.nidulans:
rodA (467 bp)
xlnA (749 bp)
sho1 (934 bp)
npc2 (627 bp)
- Purification of PCR products
- BP reaction into pDONR vector
- Transformation into DH5α
⇒ Trafo failed
May 26th, week 3
C. albicans & C. glabrata
- Transformation of BP reaction into fresh competent cells (50 µl+ rest)
- Transformation Efficienty test for supercompetent cells with iGEM kit (control with fresh competent cells)
⇒ Did not work → Kit is not useful !
- Transformation of all BP reactions worked, except for als1 and als3
- 3 colonies from each transformation were picked and dissolved in 10 µl H2O. 5µl of this were used for cracking and 5 µl to incubate a 4 ml LB culture over day.
Agarose Gels:
Samples A (from each plasmid):
Samples B (from each plasmid):
Samples C (from each plasmid):
⇒ Plasmids can be found in almost all cultures → one positive culture for each plasmid was chosen and centrifuged (5 min, 5000rpm)
- PCR for als1 and als3 were repeated:
⇒1+3 purification (eluted in 70 µl H2O)
⇒ Nano Drop:
1 8,8 ng/µl
2 24,3 ng/µl
→ PCR product from first PCR for als1 and from second PCR for als3 were used for the BP reaction (+Trafo)
- Plasmid isolation of the samples from C. albicans, C. glabrata and A. Fumigates (see protocol) (o/n cultures)
- NanoDrop results:
A. fumigatus & A. nidulans
- BP reactions of PCR products into pDONR207 and transformation into DH5α
- Transformation into E.coli DH5α cells
⇒ Trafo worked
- Cracking of e different colonies from each transformation (Agarose gel pictures see Candida notebook)
- 4 ml LB + gentamycin inoculated with positive clones
- Plasmid isolation (pIGA plasmids)
- NanoDrop:
S. cerevisiae
- Amplification of the genes of interest via PCR:
⇒ S. cerevisiae:
CWP1 (700 bp)
CWP (300 bp)
TIR1 (800 bp)
SED1 (1000 bp)
TIR4 (1500bp)
MID2 (1200 bp)
Agarose Gel (1%):
⇒ 1,2,3,4,7,8,11,12 worked; 9 + 10 will be repeated because of the two separate bands
- Purification of right fragments
- NanoDrop:
- BP reaction of CWP1, CWP2, SED1, MID2
- Transformation into DH5α
⇒ Trafo worked
- Repeat PCR for TIR1 and TIR4 fragments → no bands in agarose gel
May 19th, week 2
C. albicans & C. glabrata
- Amplification of the genes of interest via PCR
⇒ C. albicans: mp65, als1, tos1, sim1, als3
⇒ C. glabrata: ssr1, pir4, scw4, pir3, utr2
⇒ Annealing temperature: 56 °C
- Agarose Gel (1%):
⇒ Band 5 (als3) is a little bit weak → but PCR product is also purified and further used
⇒ Purification of the PCR products with the Nucleo Spin® Gel and PCR Clean-up kit from Macherey-Nagel
⇒ Nano Drop Values were not useful
- Preparation of supercompetent E.coli DH5α cells (see protocol)
- BP reaction of all genes (see protocol)
A. fumigatus & A. nidulans
- Amplification of the genes of interest via PCR (→ Annealing Temperature)
⇒ A. fumigatus:
crf2 full (mature peptide AA: 21-416) (58°C)
crf2 act (active side AA: 21-190) (56°C)
bglE no ( no GlcNAc outside AA: 594-908) (58°C)
ecm33 ( active protein AA: 20-372) (58°C)
sun1 (sequence between GlcNAc AA: 80-1133) (58°C)
rodA (AA: 19 – 159) (56°C)
prm1 (extracellular AA: 419-603) (58°C)
crf1 (active side before GlcNac AA: 20-310) (58°C)
Agarose gel (1%):
⇒ Purification of the PCR products with the Nucleo Spin® Gel and PCR Clean-up kit from Macherey-Nagel
May 12th, week 1
C. albicans & C. glabrata
- Test of supercompetent E.coli cells with the transformation efficiency kit (from iGEM)
⇒ Did not work! No colonies
- Got cDNA of Candida albicans and Candida glabrata from Oliver Baders Lab:
Candida albicans strain SCS314
Candida glabrata strain CBS138
⇒ Both high concentrations : dilution (10 µl DNA + 990 µl H2O)
A. fumigatus & A. nidulans
- Preparation of competent cells E.coli DB3.1 (mutated gyrase, resistant against ccdB) with CaCl2 (see Protocol for fresh comp. cells)
- Transformation with pDONR207 and pDEST (Plating of 50µl and rest on LB+ Cm plates, incubation over night at 37°C)
⇒ Trafo worked! Single colonies in 4 ml LB + 4µl Cm
⇒ Also saving of the strains on plates
- Plasmid isolation with M&N Kit, Elution in 70µl dH2O
- Nano Drop: