Team:Goettingen/notebook wetlab

October 13th, week 23

A. fumigatus & A. nidulans

A. fumigatus
- Results of robot Y2H:
(red color indicates a positive interaction, a blue colony. The grey color means that the result is not clear and it could possibly be a positive colony)

      ⇒ Growth and blue color indicates that there is an interaction between surface protein and the peptag.
      ⇒ to verify the interaction we will select on the peptag-plasmids and isolate them and transfer them into strep-vector (pGP172) for over expression in BL21(DE3)
      ⇒ the isolated protein is then used for in vivo studies

C. albicans & C. glabrata

- Results of the robot Y2H (see Aspergillus)
- New in vivo experiments with Ssr1

October 6th, week 22

A. fumigatus & A. nidulans

A. fumigatus
- Continue the robot Y2H: replicate colonies from selection medium to selection medium with X-α-Gal
- First in vivo experiment with the interacting peptide of Sun1 from Aspergillus fumigatus

A.nidulans
- Continue the robot Y2H (plate on to SC-LWH + 3mM 3-AT plates and X-α-gal plates)

C. albicans & C. glabrata

- Continue the robot Y2H (plate on to SC-LWH + 3mM 3-AT plates and X-α-gal plates)
- In vivo test with Candida glabrata ssr1 – k.o. strain → no fluorescents, due to too low protein concentrations (only worked for Sun1 from Aspergillus fumigatus)

S. cerevisiae

- Continue the robot Y2H (plate on to SC-LWH + 3mM 3-AT plates and X-α-gal plates)

T. gondii

- Continue the robot Y2H (plate on to SC-LWH + 3mM 3-AT plates and X-α-gal plates)

September 29th, week 21

A. fumigatus & A. nidulans

A. fumigatus
- Robot Y2H to confirm interaction
      ⇒ Tested samples: AIG001, 02, 03, 04, 05, 06, 09, 10, 12, 13, 14, 15, 17, 19
- Grow AIG strains and isolated interaction partners (YIG strains) in selection medium over night at 30 °C
- Pipet grown colonies in 96 well plate (200 µl each)
- Preparation of robo-agar-plates
- Use AIG 96 well to stamp to -trp plates, YIG for - leu plates and incubate for 2 days at 30 °C
- Mating of grown colonies on YPAD plates
- Replicate colonies from YPAD to selection plates
- Replicate colonies from selction to selection medium

A.nidulans
- Robot Y2H to confirm interaction.

C. albicans & C. glabrata

- Robot Y2H to confirm interaction (see Aspergillus)

S. cerevisiae

- Inoculation of AIG021 + 22 and YIG021 + 22 for robot Y2H

T. gondii

- Inoculation of AIG025and YIG025 for robot Y2H

September 22nd, week 20

C. albicans & C. glabrata

- Dialysis of purificated proteins (see Aspergillus)
- Preparation for robot Y2H

A. fumigatus & A. nidulans

A. fumigatus
- Contamination on robot plates → Again
- Prepare 10x PBS buffer and medium for in vivo tests
- Boil dialysis tubes for 10 minutes and add EDTA. Cool them down and fill in proteins for dialysis. Dialyze overnight at 4 °C.
- Streak out AIG04 on SC - Trp, YIG04.1 and YIG04.2 on SC - Leu
- Prepare robot plates the robot Y2H

September 15th, week 19

C. albicans & C. glabrata

- Purification of the peptides 3, 4.1, 4.2, 5 and 13
      • 1 L LB inoculated at an OD600 of 0.2 → grown to 0.8, then expression is induced with 1mM IPTG
      • After 3h: centrifuged at 5000rpm for 15 min at 4°C
      • After solving in Buffer W the cells are disrupted by French press and centrifuged for 1h at 35.000 rpm and 4 °C
      • The proteins are extracted via Strep-Tactin® columns
      • Samples were loaded onto an 12% SDS Gel

SDS Gels (12%)
Strep 3:
⇒ There should be a clear band in the elutions steps for ~ 20 -22 kDa; for strep 3 is no such band

Strep 4.1:
⇒ No band of ~20-22 kDa in the elution steps for strep 4

Strep 4.2:
⇒ The right band can be seen in the elution steps, but it is not as strong as it should be

Strep 5:
⇒ The enlarged band in the elution steps has a size of ~ 15 kDa, which seems to be a little small for our peptide

Strep 13:
⇒ For strep 13 the right bands of ~20-22 kDa can be seen in all lines, especially in the elution steps

A. fumigatus & A. nidulans

A. fumigatus
- Starting Seamless cloning mix, incubate at RT for 30 minutes, 4 minutes on ice and then E. coli transformation into TOP10 strain. Plating 200 µl and rest on cat plates.
- Determination of OD600 of strep cultures and inoculate 1 L LB + Amp to an OD of 0.2 and let them grow to an OD of 0.8 (approx. 1 h). Induction with 1 ml of 1 M IPTG and incubation at 180 rpm and 37 °C for 3 hours. Centrifugation in autoclaved bottles for 15 minutes at 5 krpm at 4 °C. Freeze pellets in -20 °C overnight.
- Preparing robot plates and distribute 200 µl of each cell culture
- Run spotting program on the robot. And incubate at 30 °C
- Pre-cool french press stuff in ice water. Pre-cool Buffer W and tips and columns in cold room at 4 °C. Resuspend each pellet in 15 ml ice cold Buffer W. Run french press as in the protocol. Centrifuge the disrupted cells at 4 °C for 1 h and 35.000 krpm.
- Prepare columns, Buffer E, Buffer R and pack each column with 1 ml streptacin 50 %.
- Run the supernatant from UZ over the columns. Safe rest in falcon tubes from not purified, flow through. Save wash step 1- 4 elution step 1- 4 in labeled 1.5 ml Ecups.
- Prepare 4 SDS gels 12.0 % (Gels see Candida)
- Calculation of protein amount in CE and FT with Bradford.
- Y2H with robot to confirm interactions

S. cerevisiae

- Repeat trafo in Y187
      ⇒ Trafo worked

T. gondii

- Repeat trafo in Y187
      ⇒ Trafo worked

September 8th, week 18

C. albicans & C. glabrata

- PCR for sequencing on strep-tag plasmids
- Purification and sequencing
      ⇒ Sequencing looks ok
- Test expression for peptides 3, 4.1, 4.2, 5, 13
SDS gels (12%)
Peptide 3:
Peptides 13 and 4.1:
Peptides 4.2 and 5:
- Test expression looks good; nothing in negative control (NK)
- super-competent cells with iGEM protocol → see Aspergillus

A. fumigatus & A. nidulans

A. fumigatus
- PCR with T7 primers for sequencing of 6 plasmid inserts (seqlab) → purification
- NanoDrop:
- Pick colonies from strep plates inoculate over night culture. Inoculation over day culture to OD600 0.3. Prepare test expression
- Ligation of cut clean pGP172 (11ng/µl) with cc5, 9, 10 ,15
- Follow blunt end-ligation protocol: 20 µl sample included 2 µl PEG4000 1 µl T4 ligase
- Preparation of final robot Y2H
- Transformation into E. coli DH5α
- Sequences of Seqlab looks ok
- Test expression of 3, 4, 13
- Samples from test expression were filled up with 15 µl of SDS loading dye and boiled for 15 min at 95 °C. Loaded to SDS 12% PAGE. (Gels see Candida)
- Prepare comp. cells from TOP10 strain. Overnight culture was grown at 18°C for 19h OD600=0.395 → 1:1 dilution with 250 ml SOB for 1 h incubation at 18 °C. OD600 = 0.32, putted on ice and filled into prechilled 50 ml flasks. Followed the protocol.
      ⇒ Test the competence → worked very well!

A.nidulans
- prepare robot screening for 2st Y2H with the isolated plasmids out of the Y187 transformation
- 2st Y2H screening on plates - failed

S. cerevisiae

- Pick colonies from E. coli plates and isolate plasmids
- Transformation of yeast (Y187)
      ⇒ Trafo failed

T. gondii

- Pick colonies from E. coli plates and isolate plasmids
- Transformation of yeast (Y187)
      ⇒ Trafo failed

September 1st, week 17

C. albicans & C. glabrata

- New ligation of 4.1 (ssr1 interacting peptide) (see Aspergillus)
      ⇒ Test via PCR, if insert is in the vector (bigger fragment) → see Aspergillus

- Sequencing: again 10 + 15 (+ BP1)
      ⇒ Results: 10 and 15 are exactly the same interacting peptide

A. fumigatus & A. nidulans

A. fumigatus
- Measure DNA conc. pGP172 → 350ng/µl
- Use 1.000 ng for restriction. 16 µl mixture 4 µl buffer (Tango) 2 µl Sma1 incubation for 120 minutes at 30 °C. Then add 2 µl of BamH1 for more 2 h. dephosphorylation with SAP for additional 30 minutes.
- Measure OD of o/N culture Y187 (5.4) and AH109 (18.4). Inoculation of 50 ml YPAD 0.54 ml and 1.89 ml
- AH109 0.7 OD600 follow long term storage protocol. Freeze at -80°C there was no sorbitol inside
- purification of pGP172 - nanoDrop: 50ng
- Ligation over night at 16 °C
- cut pGP172 + fragments cc_number
- Inoculation of AH109/Y187 for storage cultures
- PCR with PIGY9 and 10 with IG099 and IG1003
- Measure OD600 from o/N cultures; AH109 [12.9] and Y187 [12.1]. →use 0.78 ml and 0.82 ml of the strains to inoculate to an OD600 of 0.2. After 3 hours follow long term storage protocol
- Purification of PCR product from 9 and 10
- Cut pIGY9 and 10 with Hpa1 and BamH1
Agarose Gel (1%) - Prep. of pGP172cut and clean: Sma1 and BamH1 - SAP 50 µl sample and purification
- Transformation of all ligations → nothing on plates
- Inoculation of BL21blue and pGP172.
- Restriction of pGP172 again for ligation of inserts. 1:1 ratio, Ligation with three inserts 3, 4.1 and 15.
- Transformation into DH5α.
- Growth on plates: Pick 10 colonies from each transformation plates grow over day 37°C with respective antibiotics and start plasmid isolation - stopped at 2-prop step over night [also pGP172 3x purification].
- New Oligos: Check PCR with T7p and T7t (52°) for strep
- PCR (12,5µl samples) with strep-4.1 and strep-NK(15/3?)
Agarose Gels (1%):       ⇒ Bands in 3, 4, 5, 7, 8, 9 are positive
      ⇒ Bands in 4 and 8 are positive
- Inoculations from overnight culture a 500 ml flask with 50 ml LB and shake it till OD600 0.35 and follow fast comp. cell protocol. Use 2 µl from each plasmid and incubation over 2 days at 37°C.

S. cerevisiae

- Spot positive clones on SC-LWH plates with 3mM 3-AT
- Wash cells from plates and isolate plasmids
- Transformation into DH5α

T. gondii

- 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

C. albicans & C. glabrata

- 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→?
- New test expression (see Aspergillus)

A. fumigatus & A. nidulans

A. fumigatus
- centrifuged 5 min at 13 krpm - freeze at -20 °C
- Preparation of Amp and Cm plates, streak out of used strains on Amp-plates
- SDS-gels prepared and stored in PLP 1x buffer at 4 °C
- staining of sds gel → empty
- PCR with fragments (3, 13, 15) (Agarose gel see Candida)
      ⇒ Purification of: 13.3 15 3.1A 3.2A 3.3A 3.1B 3.2B
- purified PCR products for restriction, ligation and transformation
- Preparation of 10 x PLP buffer and new SDS-gels
- Loading of 5 µl sample onto the gels→ expected size empty ~11.5 kDa
- New test expression at 30 °C and test also supernatant.
- Inoculation over night cultures of desired strains.
- Toxicity cannot be the case, Protein G subunit backbone was shown to hide this
- Streak out DB3.1 strains containing pDONR and pDEST
- Inoculation of over day cultures to OD600 of 0.1. Grow for 90 minutes at 30 °C
- Measure OD600 and take sample (safe also the supernatant)
- Add IPTG and take samples after 1, 2, 3 hours.
- Follow test expression protocol
- PCR with strep-tagged proteins T7 promotor / terminator
Agarose Gel (1%):
- cut again pGP172 (520 ng and 1080 ng) with Sma1 and BamH1 (Tango Buffer for 2 h)
- pick 5 different colonies from strep tagged inserts into DH5α → colony PCR
      ⇒ false inserts ☹
- Transformation of ligation into DH5α

A.nidulans
- Repeat yest transformation in Y187 with the genes rodA and npc2
- isolate plasmids

S. cerevisiae

- Y2H in semi solid media with the two genes

T. gondii

- Y2H in semi solid media

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→?

A. fumigatus & A. nidulans

A. fumigatus
- PCR from diff. plasmids for prey amplification 52,5 °C (Agarose Gel see Candida)
      ⇒ Worked for all
- Purification of prey pcr products, restriction with HpaI and BamH1
- restriction of 0.1 µl (14500 ng/µl) pGP172 with Sma1 and BamH1 o/n 16 °C
- ligation of cutted cleaned PCR prey products with pGP172 (10 ng/µl); 4 µl of each with 1 µl T4 Ligase in 20 µl sample
- PCR (100 µl vol) with sample 5 and 4.1 to increase amount of product
Agarose gel(1%)
- Transformation of strep-prey plasmids worked. over day cultures in LB at 42 °C for plasmid isolation and colony PCR
- Inoculation of E. coli BL21blue for fresh comp cell to transform strep plasmids
- pGP172 o/N 16 °C restriction compared with 0,1 µl and 1 µl non cut plasmid
- nothing in colony PCR
- pellets from over day cultures
- Plasmid isolation
NanoDrop:
- PCR with T7 promotor and terminator primer on plasmids (Agarose gel see Candida)
- LB medium, BL21 blue competent cells, transformation with plasmids
- Transformation of BL21blue with the plasmids worked
- Inoculation of 10 ml LB medium incubation for 2 h at 37 °C
- Take samples before induction and after 1 - 2 - 3 h [ 100/OD600 ]
- centrifuged 5 min at 13 krpm → freeze at -20°C
- Preparation of Amp and Cm plates, streak out of used strains on Amp-plates
- SDS-gels prepared and stored in 1x PLP buffer at 4 °C

A.nidulans
- Repeat transformation of isolated plasmids out of the 1st Y2H experiment in E. coli DH5α
- Plasmid isolation out of E. coli DH5α
- NanoDrop:
- Repeat transformation in Y187 on SC-Leu medium - failed

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

A. fumigatus & A. nidulans

A.nidulans
- Repeat transformation in Y187 on SC-Leu medium - failed
- Repeat transformation in Y187 on SC-Leu medium- failed
- running out of isolated plasmids out of E. coli DH5α for Yeast transformation

August 4th, week 13

A. fumigatus & A. nidulans

A. fumigatus
- O/N cultures from E. coli prey colonies to isolate plasmids again for restriction and ligation into pGP172

A.nidulans
- Transformation in E. coli DH5α
- Plasmid isolation out of E. coli DH5α
- Transformation in Y187 - failed

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

T. gondii

- Yeast transformation (AH109)

July 21st, 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

A. fumigatus & A. nidulans

A. fumigatus
- pick colonies = o/n cultures for plasmid isolation
- plasmid- prep. of pGP172 acc. to the protocol

A.nidulans
- 1st Y2H experiment with the genes rodA, xlnA, npc2, slno1 in semi solid medium - worked only for the plasmids pIGA17 (rodA) and pIGA19 (npc2)
- after growing the yeast colonies are washed from plates
- plasmid isolation

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:

A. fumigatus & A. nidulans

A. fumigatus
- Test of mated colonies from robot Y2H on X- α-gal plates
- 3rd Y2H: after spotting, wash colonies from plates and plasmid isolation

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
Titerplates:
- Robot Y2H with 04, 05, 09, 10, 14 (see Candida)
- Transformation of 02, 06, 15, 16 pool into DH5α
      ⇒ Only grown with single colonies → again with different concentrations

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

T. gondii

- Repeat LR reaction and transformation into DH5α
      ⇒ Trafo worked
- Cracking of E. coli cells → right plasmid in cells
- Plasmid isolation (2528,7 ng/µl)

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(Example)

- 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:

T. gondii

- LR reaction and transformation into DH5α
      ⇒ Trafo didn’t work

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

T. gondii

- Repeat BP reaction and transformation into DH5α
      ⇒ Trafo worked
- E. coli cracking
Agarose Gel (1%):
- Plasmid isolation of ama1 (pIGA25); NanoDrop: 1308,3 ng/µl

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 2nd, 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

T. gondii

- Amplification of the gene of interest via PCR:
      ⇒ Toxoplasma: ama1 (Annealing temperature) 54°C
Agarose Gel(1%):
      ⇒ Purification of fragment. NanoDrop → 157,6 ng/µl

- BP reaction and transformation into DH5α
      ⇒ Trafo didn’t work

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: