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Team:Goettingen/notebook wetlab/bait team
From 2014.igem.org
Notebook
October
September
August 18th, week15
Bait team
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→?
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
August 11th, week14
Bait team
- 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
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
August 4th, week13
Bait team
On the vacation.
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
July 28th, week12
Bait team
- 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
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
July 21th, week11
Bait team
- 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
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
July 14th, week10
Bait team
- 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:
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
July 7th, week9
Bait team
- 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
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
June 30th, week8
Bait team
- 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)
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
June 23rd, week7
Bait team
- 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
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
June 16th, week6
Bait team
- 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
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
June 9th, week5
Bait team
- 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:
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
June 2rd, week4
Bait team
- 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
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
May 26th, week3
Bait team
- 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:
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
May 19th, week2
Bait team
- 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)
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
May 12th, week1
Bait team
- 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)
Prey team
It hasn't started yet.
Biobrick team
It hasn't started yet.
