Journal
July
Week 4: july 22 - 28
- Introduction given by our lab instructors
- General techniques: plasmid/PCR purification, inoculation, gel extraction, restriction & ligation.
- Safety and waste disposal training
- Introduction to CloneManager
- General preparations: sterile mQ, sterile eppendorf
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August
Week 1: july 29 - august 4
Week 2: august 5 - 11
- Experiment 1
- Inoculate E. coli DH5a + pTGD-ccdA-Pmb1GFP-CmFRT
- Purify plasmid pTGD-ccdA-Pmb1GFP-CmFRT using Qiagen spin mini kit: Nanodrop -- 362.4 ng/µL
- Preparative Restriction Digest (RD) of purified plasmid: BspHI & BstAPI: expected fragments of 5541bp and 903 bp
- Gel purify RD-fragment of 5541 bp using Qiagen Qiaquick gel extraction kit: Nanodrop -- 70.4 ng/µL
- PCR on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT with MDM588 & MDM589
->HiFi PCR using Primestar polymerase. Analytical gel: negative
->PCR using Q5 polymerase. Analytical gel: negative
->PCR using Roche. Analytical gel: negative
->Touchdown PCR using Q5 polymerase. Analytical gel: negative
- Control plasmid pTGD-ccdA-PMbAFGP-CmFRT: RD with AclI. Expected fragments: 758 bp, 1730 bp and 3956 bp. Analytical gel: negative --> Problem with plasmid pTGD-ccdA-PMbAFGP-CmFRT
- Transformation: Knock in with lineair back-up DNA by electroporation. Incubation of the transformed cells and transfer the culture on a Cm plate.
- Colony PCR on 48 colonies using crimson taq polymerase with two primer pairs: MDM0141/MDM0010 and MDM0046/MDM123. Expected fragments: 550bp & 2450 bp. Analytic gel: 4 positives
- Colony PCR on 4 positive and 4 negative colonies using crimson taq polymerase with out primers: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: negative .
- 2 Colony PCR on 4 positive and 4 negative colonies using Taq and Phire polymerase with out primers: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: negative .
- Colony PCR on 4 positive and 4 negative colonies using Emerald polymerase with out primers: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: 1 positive: colonie 35 .
- Experiment 2
- Inoculate E. coli DH5a + p5SpFRT-T7ccdB
Inoculate E. coli DH5a + p10SpFRT-T7ccdB
Inoculate E. coli DH5a + p20SpFRT-T7ccdB
- Purify plasmids using Qiagen spin mini kit: nanodrop
p5SpFRT-T7ccdB: 119,6 ng/µl
p10SpFRT-T7ccdB: 156,3 ng/µl
p20SpFRT-T7ccdB: 392,9 ng/µl
- CcdB operon:
-> HiFi PCR of plasmids p5SpFRT-T7ccdB, p10SpFRT-T7ccdB and p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify ccdB operon
-> Purification of CcdB operon PCR fragment using Qiagen Qiaquick PCR purification kit and checked on analytical gel (expected fragment of 2300 bp): nanodrop
p5: 174,6 ng/µl
p10: 24,5 ng/µl (consistent with small band on the analytival gel)
p20: 104,3 ng/µl
-> Redo of inoculation E. coli DH5a + p5SpFRT-T7ccdB, E. coli DH5a + p10SpFRT-T7ccdB and E. coli DH5a + p20SpFRT-T7ccdB
-> Redo of purification of plasmids p5SpFRT-T7ccdB, p10SpFRT-T7ccdB and p20SpFRT-T7ccdB using Qiagen Spin minikit: nanodrop
p5SpFRT-T7ccdB: 21,1 ng/µl
p10SpFRT-T7ccdB: 25,1 ng/µl
p20SpFRT-T7ccdB: 24,6 ng/µl
- Vectors pSB4A5 (plate 5, well 5I), pSB3T5 (plate 2, well 8D) and pSB6A1 (plate 2, well 2L):
-> Resuspend plasmids from the iGEM kit
-> Transform in E. coli Top10 subcloning cells using elektroporation
-> Plate pSB4A5 and pSB6A1 on ampicillin plate and pSB3T5 on tetracyclin
and grow overnight at 37°C
-> Plates with transformants: pSB6A1 lots of colonies, pSB3T5 sufficient colonies and pSB4A5 no colonies
- Vectors pSB4A5 (plate 5, well 5I):
-> Resuspend plasmids from the iGEM kit
-> Transform in E. coli Top10 subcloning cells using elektroporation
-> Plate on ampicillin plate and grow overnight at 37°C (1 plate 150 µl, 1 plate 50 µl)
-> Plates with transformants: pSB4A5 no colonies
- Inoculation colonies of pSB3T5 and pSB6A1 -> at the end of the day replaced from 37°C to 30°C
- Vector pSB4A5 (plate 5, well 1I and plate 2, well 2J as backup) and pSB6A1 (plate 5, well 1K as backup):
-> Resuspend plasmid pSB4A5 and pSB6A1 from the iGEM kit
-> Transform in E. coli Top10 subcloning cells (heat shock)
-> Plate transformation on ampicillin plate and grow overnight at 37°C (3 plates: 10-2,10-1 and 100)
-> Plates with transformants: pSB4A5 (plate 2, well 2J) colonies, pSB4A5 (plate 5, well 1I) no colonies and pSB6A1 (plate 2, 2L) few colonies
- Inoculate pSB3T5 and pSB6A1 again and in warm chamber (37°C).
- Inoculation colonies of pSB4A5 (plate 2, well 2J) and pSB6A1 (plate 2, well 2L) (2 times: one for further work and one for cryovials)
- Purification of plasmids pSB3T5, pSB6A1 and pSB4A5
- Generation of restriction digest fragments of T7-ccdB insert (from p5SpFRT-T7ccdB and p20SpFRT-T7ccdB) and vector (pSB3T5 (2x), pSB4A5 (from plate 2, well 2J) and pSB6A1 (1x from plate 5, well 1K and 1x from plate 2, well 2L) with XbaI and PstI-HF (gel purify RD-fragments using Qiagen Qiaquick gel extraction kit (expected fragment of 2157 bp for T7-ccdB, 3229 bp for pSB3T5, 3372 bp for pSB4A5 and 3999 bp for pSB6A1): Nanodrop
p5: 43,7 ng/µl
p20: 24,2 ng/µl
3T5 (inoculation 1): 23,5 ng/µl
3T5 (inoculation 2): 23, ng/µl
4A5: 40,5 ng/µl
6A1 (plate 2, 2L): 40,1 ng/µl
6A1 (plate 5, 1K): 30,4 ng/µl
- Ligation of T7ccdB from p5SpFRT-T7ccdB and pSB6A1 (1x from plate 5, well 1K and 1x from plate 2, well 2L), p20SpFRT-T7ccdB and pSB3T5, p20SpFRT-T7ccdB and pSB4A5 (from plate 2, well 2J) (3(insert)/1(plasmid) ratio)
- Transformation in E.coli Top10 (heat shock)
- Plate transformation on ampicillin (pSB4A5 and pSB6A1) and tetracycline plate (pSB3T5) and grow overnight at 37°C (2 plates: 10-2, 10-1)
-> Plates with transformants: no colonies
- New transformation in E.coli Top10 from pSB4A5 and pSB6A1 using heat shock
-> Plates with transformants: pSB6A1 2 colonies, pSB4A5 few colonies
- New ligation with RD-fragments created before (vectors pSB3T5, pSB4A5, pSB6A1 (plate 5, well 1K) and insert T7-ccdB created in experiment 6), at 22,5°C o/n)
- CcdB operon:
-> HiFi PCR of plasmids p5SpFRT-T7ccdB, p10SpFRT-T7ccdB and p20SpFRT-T7ccdB with MDM0586 & MDM0587 to amplify ccdB operon
- Experiment 6
- CcdB operon:
-> HiFi PCR of plasmids p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify ccdB operon
nanodrop: 301,5 ng/µl
- Vector pSB1C3 (plate 1, well 23O):
-> Resuspend plasmids from the iGEM kit
-> Transform pSB1C3 in E. coli Top10 subcloning cells, using heat shock (42°C)
-> Plate on chloramphenicol plate and grow overnight at 37°C
-> Inoculate E. coli Top10 + pSB1C3
-> Purify plasmids using Qiagen Qiaprep Spin minikit: nanodrop: 200,2 ng/µl
- Restriction of CcdB operon and pSB1C3 with XbaI and PstI
-> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
Restriction digest of T7-ccdB: 16.9 ng/µl
Restriction digest of pSB1C3: 12.3 ng/µl
- Ligation of CcdB operon and pSB1C3
- Transformation in E. coli Top10 subcloning cells, using heat shock (42°C)
-> Plate on chloramphenicol plate and grow overnight at 37°C: negative
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Week 3: august 12 - 18
- Experiment 1
- Inoculation positive colonie on cm plate.
- Colony PCR on 8 kolonies using emerald polymerase with out primers: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: positive: 5 & 8 .
- Experiment 2
- PCR purification of CcdB operon: nanodrop
p5: 54,8 ng/µl
p10: 65,8 ng/µl
p20: 21,1 ng/µl
- cPCR with Taq polymerase of colonies pSB6A1 (primers: MDM0096 and CLG0019) and pSB4A5 (primers: MDM0095 and CLG0019): negative
- New transformation with pSB3T5, pSB4A5 and pSB6A1 using heat shock in E.coli DH5a
-> Plate on normal plates and glucose plates (100 ml 2% glucose, to avoid leaky expressing of ccdB)
-> Plates with transformants: pSB6A1 some colonies, pSB4A5 some colonies and pSB3T5 no colonies
- Gel elektroforesis on RD-fragments from plasmids: show expected fragments
- cPCR on the pSB4A5 (primers: MDM0095 and CLG0019) and pSB6A1 (primers: MDM0096 and CLG0019) colonies with Taq polymerase: one positive colony of pSB6A1-T7ccdB (fragment of 437 bp in lane 14)
- inoculation of positive pSB6A1-T7ccdB colony from back up plate (3x: one for sequencing, 1 for cryovial and 1 for experiment 3)
- Plate transformation mixtures again on glucose plates and grow overnight at 30°C
->Plates with transformants: pSB4A5 colonies, pSB3T5 no colonies
- cPCR on pSB4A5 colonies (primers: MDM0095 and CLG0019) with Taq polymerase: negative
- New ligation with rest of RD-fragments of plasmids pSB3T5 (2 times) and pSB4A5 and CcdB from experiment 6 and transformation in DH5a using heat shock
- Due to lack of success: start from the beginning.
- Inoculation of pSB3T5, pSB4A5, p5SpFRT-T7ccdB, p10SpFRT-T7ccdB and p10SpFRT-T7ccdB from cryovials.
- Also resuspend pSB3T5 (plate 5, well 7C) from iGEM kit, transform in E. coli DH5a using heat shock, plate on tetracycline plates and incubate overnight at 37°C
-> no colonies
-> transform again in DH5a using heat shock
-> again no colonies
- Purification of plasmids using Qiagen spin minikit: concentrations deduced from gel
p5SpFRT-T7ccdB: +/- 40 ng/µl
p10SpFRT-T7ccdB: +/- 40 ng/µl
p20SpFRT-T7ccdB: +/- 40 ng/µl
pSB3T4: +/- 250 ng/µl
pSB4A5 (1): +/- 400 ng/µl
pSB4A5 (2): +/- 350 ng/µl
- PCR on CcdB operon using Q5 polymerase (primers: MDM0586_Fw-Trc-ccdB-G00000 and MDM0587_Rv-Trc-ccdB-G00001)
- cPCR on pSB3T5 colonies from old plates using Taq polymerase (primers: MDM0602 and CLG0019, expected fragment of 503 bp): negative
- Restriction of ccdB operon, pSB3T5 and pSB4A5 using PstI-HF and XbaI (gel purify RD-fragments using Qiagen Qiaquick gel extraction kit (expected fragment of 2157 bp for T7-ccdB, 3229 bp for pSB3T5 and 3372 bp for pSB4A5): concentrations deduced from gel
- Ligation of pSB3T5 and T7ccdB (from p5SpFRT-T7ccdB), pSB4A5 and T7ccdB (from p5SpFRT-T7ccdB) and pSB4A5 and T7ccdB (from p20SpFRT-T7ccdB) with T4 DNA ligase at 16°C overnight
- Transformation of ligation mixtures in DH5a using heat shock
-> no colonies
- Experiment 3
- Inoculation KI-strain 5 & 8 from experiment 1
- Transformation of pSB6A1 - T7ccdB in KI-strains 1,5 and 8
- Colony PCR on kolonies from transformed strains using emerald polymerase with out primers to check KI: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: positive
- Colony PCR on kolonies from transformed strains using emerald polymerase with MDM0096 & CLG0019 to check if plasmid is present. Expected fragment: 437 bp. Analytic gel: positive
- Experiment 6
- New transformation in E. coli Top10 subcloning cells, using heat shock (42°C)
-> Plate on chloramphenicol plate and grow overnight at 37°C: negative
- CcdB operon: HiFi PCR of plasmids p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify CcdB operon
- New restriction of CcdB operon and pSB1C3 with XbaI and PstI
-> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
Restriction Digest of T7-ccdB: 16.6 ng/µl
Restriction Digest of pSB1C3: 17.7 ng/µl
- Ligation of CcdB operon and pSB1C3: overnight at 16°C
- Transformation in E. coli DH5a, using heat shock (42°C)
-> Plate on chloramphenicol + glucose plate and grow overnight at 30°C: negative
- Restriction of CcdB operon and pSB1C3 with XbaI and PstI
Because a preparative gel purification cause low concentrations, we will use DpnI
-> Purification by using minElute reaction cleanup kit
- Control of restriction fragments: positive, so we hope that ligation will succeed
- Ligation of CcdB operon and pSB1C3: overnight at 16°C
- Transformation in E. coli DH5a, using heat shock (42°C)
-> Plate on chloramphenicol plate and grow overnight at 37°C: negative
(it does not work, maby our transformation is not succeed)
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Week 4: august 19 - 25
- Experiment 2
- pSB6A1-T7ccdB sent for sequencing with primers MDM0096 (A471969), MDM0060 (A471682) and MDM0039 (A471681)
-> correct sequence
- Transform ligation mixtures again in DH5a, using heat shock (42°C),
-> no colonies
- PCR on CcdB operon with Q5 polymerase (primers: MDM0586_Fw-Trc-ccdB-G00000 & MDM0587_Rv-Trc-ccdB-G00001) and PCR purification using Qiagen Qiaquick PCR purification kit: nanodrop
p5: 15.2 ng/µl
p10: 18.6 ng/µl
p20: 43.5 ng/µl
- Inoculation of pSB3T5 and pSB4A5 from cryovials and plasmid purification using Qiagen Spin minikit: nanodrop
pSB3T5: 75.7 ng/µl
pSB4A5: 190.0 ng/µl
- Restriction of CcdB operon, pSB3T5, pSB4A5 and the pSB6A1-T7ccdB transformant with PstI-HF and XbaI (gel purify restriction digest-fragments using Qiagen Qiaquick gel extraction kit (expected fragment of 2157 bp for T7-ccdB, 3229 bp for pSB3T5 and 3372 bp for pSB4A5)
- Ligation
- Transformation in E. coli DH5a
-> no colonies
-> plate transformants on plates with a lower antibiotic concentration (50% lower)
-> no colonies
- Design primers for Gibson assembly
- Experiment 3
- Colony PCR with higher annealing temperature to minimize false priming on kolonies from transformed strains using emerald polymerase with out primers to check KI: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: positive
- HiFi PCR using Q5 polymerase with two primer pairs generating overlapping fragments of the ccdA.gfp.cat construct : MDM0046 (out) & MDM0123: 2130 bp and MEMO1237 & MDM0010 (out): 3400 bp. Analytic gel: negative
- Transformation of backup plasmids:p5SpFRT-T7ccdB, p10SpFRT-T7ccdB, p20SpFRT-T7ccdB in backup KI-strain
- Experiment 4
- Lysate preparation:
-> Dilution of o/n culture of BW26547 in 5 mL LB+MgCL2+CaCl2+glucose
-> Add phage lysate (10,20,40 & 80 µL)
-> Wait untill lysis visible
- Perform CIChE with 8.1 + pSB6A1 & MDM + pSB6A1 : 0,0mM - 0,05mM IPTG
- Transduction of CIChe strains: no positive colonies after UV-test
- Experiment 6
- New transformation in E. coli DH5a, using heat shock (42°C)
(because we are sure that the fragments were present for ligation and so will lead to succesful results)
-> Plate on chloramphenicol plate and grow overnight at 37°C: negative (it still does not work)
- Control of ligations by using PCR with primers MDM0606 and MDM0607: negative
- Ligation again of CcdB operon and pSB1C3: 15 minutes at 16°C
- Transformation in E. coli DH5a, using heat shock (42°C)
-> Plate on chloramphenicol plate and grow overnight at 37°C: negative
- CcdB operon: HiFi PCR of plasmids p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify CcdB operon
because we have no more T7ccdB fragments
- New restriction of CcdB operon and pSB1C3 with XbaI and PstI
-> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
Restriction Digest of T7-ccdB: 15.3 ng/µl
Restriction Digest of pSB1C3: 16.8 ng/µl
- Ligation of CcdB operon and pSB1C3: 1 hour at room temperature
- Transformation in E. coli DH5a, using heat shock (42°C)
-> Plate on chloramphenicol plate and grow overnight at 37°C: positive (finally)
- Colony PCR (Taq): negative
It seems that restriction-ligation does not work, thus we will use another technique, called Gibson Assembly
- Designing primers for Gibson Assembly for the insert T7ccdB and the backbone pSB1C3
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Week 5: august 26 - september 1
- Experiment 2
- Inoculation of pSB3T5, pSB4A5, pSB6A1-T7ccdB, p5SpFRT-T7ccdB, p10pFRT-T7ccdB and p20pFRT-T7ccdB and plasmid purification using Qiagen Spin minikit: nanodrop
PCR on CcdB operon using Q5 polymerase (primers: MDM0586_Fw-Trc-ccdB-G00000 and MDM0587_Rv-Trc-ccdB-G00001)
- Restriction of pSB3T5, pSB4A5 and CcdB operon with PstI-HF and XbaI (gel purify RD-fragments using Qiagen Qiaquick gel extraction kit (expected fragment of 2157 bp for T7-ccdB, 3229 bp for pSB3T5 and 3372 bp for pSB4A5): nanodrop:
-> pSB3T5: 2,7 ng/µl
-> pSB4A5: 7,8 ng/µl
- Q5 PCR of CcdB operon and plasmids (pSB3T5 and pSB4A5), using the designed Gibson primers
-> Purification of PCR-product after DpnI treatment by using minElute reaction cleanup kit
-> Control of fragments on a gel => Backbones are present, CcdB operons not
- PrimeStar PCR of CcdB operons, using the designed Gibson primers
-> Purification of PCR-product after DpnI treatment by using minElute reaction cleanup kit
-> Control of fragments on a gel => CcdB operons are still not present
- Q5 PCR of CcdB operons on a linear CcdB operon, using the designed Gibson primers
-> Purification of PCR-product, using Qiagen Qiaquick PCR purification kit,
-> Control of fragments on a gel => CcdB operon is visible
- Gibson Assembly (1h at 50°C)
- Transformation in E. coli DH5a, using heat shock (42°C)
-> Plate pSB3T5 on tetracyclin plate and grow overnight at 37°C: positive
-> Plate pSB4A5 on ampicillin plate and grow overnight at 37°C: positive
Strange phenomena: most colonies are red, let’s control it
- Colony PCR of colonies derived from Gibson Assembly: negative
- Experiment 3
- Colony PCR on MDM + pSB6A1/p5/p10/p20 using emerald polymerase with primerpairs MDM0046/MDM0123 and oMEMO 2620/MDM0010. Expected fragment: 1082bp & 239 bp. Analytic gel: positive
- Transformation of plasmids:p5SpFRT-T7ccdB, p10SpFRT-T7ccdB, p20SpFRT-T7ccdB in KI-strain (8) and back-up strain MDM.cm
- Colony PCR on MDM.cm + pSB6A1/p5/p10/p20 using emerald polymerase with primerpairs MDM0046/MDM010 to check KI. Expected fragment: 5500bp. Analytic gel: positive
- Colony PCR on MDM.cm + pSB6A1/p5/p10/p20 using emerald polymerase with primerpairs MDM0096/CGL0019 for pSB6A1 and MDM0039/MDM0060 for p5/p10/p20. Expected fragment: 437bp & 910 bp. Analytic gel: positive for p5/p10/p20
- New transformation MDM.cm +pSB6A1 and check KI + plasmid as described above. Analytic gel: positive .
- Experiment 4
- Abort CIChe with 8.1+pSB6A1 & MDM+pSB6A1 because of mutation in pSB6A1
- Lysate preparation
- CIChE with MDM + P5/P10/P20: 0mM - 0,05 mM
- Transduction: on CIChE strains described above
--> MDM grows on kan! Wrong strain
- Test lysate by performing transduction on MDM.cm with different concentrations of lysate: negative
- Antibiotic test strains: MDM grows on kan-plate
- Experiment 6
=> While waiting for the Gibson Assembly primers
- Again restriction of CcdB operon and pSB1C3 with XbaI and PstI
-> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
Restriction Digest of T7-ccdB: 10.5 ng/µl
Restriction Digest of pSB1C3: 7.2 ng/µl
- Ligation of CcdB operon and pSB1C3: overnight at 16°C
- Transformation in E. coli DH5a, using heat shock (42°C),
-> Plate on chloramphenicol plate and grow overnight at 37°C: positive
- Colony PCR (Taq) of these colonies: negative
=>After receiving the primers for Gibson Assembly
- Q5 PCR of CcdB operon and pSB1C3, using the designed Gibson primers
-> Purification of PCR-product, after DpnI treatment, by using minElute reaction cleanup kit
-> Control of fragments on a gel => Backbone is present, CcdB operon not
- PrimeStar PCR of CcdB operon, using the designed Gibson primers
-> Purification of PCR-product, after DpnI treatment, by using minElute reaction cleanup kit
-> Control of fragments on a gel => CcdB operon is not present
- Q5 PCR of CcdB operon on a linear CcdB operon, using the designed Gibson primers
-> Purification of PCR-product, using Qiagen Qiaquick PCR purification kit
-> Control of fragments on a gel => CcdB operon is visible
- Gibson Assembly (1h at 50°C)
- Transformation in E. coli DH5a, using heat shock (42°C),
-> Plate on chloramphenicol plate and grow overnight at 37°C: positive
Strange phenomena: colonies are red, let’s control it
- Colony PCR (Taq) of colonies derived from Gibson Assembly: negative
We think that some original plasmid has to be present
and the bacteria love that plasmid more than the plasmid with ccdB
Close
September
Week 1: september 2 - 8
- Experiment 2
- Restriction of CcdB operon and pSB3T5 with EcoRI and SpeI
-> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
Restriction Digest of T7-ccdB: 14.4 ng/µl
Restriction Digest of pSB3T5 : 7.2 ng/µl
- Restriction of CcdB operon and pSB4A5 with EcoRI and SpeI
-> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
Restriction Digest of T7-ccdB: 14.4 ng/µl
Restriction Digest of pSB4A5: 7.8 ng/µl
- Ligation of CcdB operon and pSB3T5 and of CcdB operon and pSB4A5: 25 minutes at room temperature
- Transformation in E. coli DH5a using heat shock
-> Plate pSB3T5 on tetracyclin plate and grow overnight at 37°C: negative
-> Plate pSB4A5 on ampicillin plate and grow overnight at 37°C: negative
- Gibson Assembly (1h at 50°C) of pSB3T5 and CcdB operon
- Transformation in E. coli Top10 subcloning cells, using electroporation (Time Constant: 4.3)
-> Plate on tetracyclin plate and grow overnight at 37°C: negative
- Gibson Assembly (1h at 50°C) of pSB4A5 and CcdB operon
- Transformation in E. coli Top10 subcloning cells, using electroporation (Time Constant: 4.4)
-> Plate on ampicillin plate and grow overnight at 37°C:negative
- Experiment 4
- CIChE with strain 8 + pSB6A1/p5/p10&p20:
--> Inoculated without antibiotic & plated out on antibiotic: 0mM - 0.5mM IPTG
--> Inoculated in antibiotic - sequentially: 0mM - 0.5mM IPTG
--> Inoculated in antibiotic - parallel: 0mM -0.5 mM IPTG
- Transduction: on CIChE strains described above --> colonies on control plate
- Experiment 5
- Test evaluation GFP with LB-medium:
-> Wild type
-> DH5a + pTGD-ccd-Pmb1GFP-CmFRT
-> Strains with GFP: 8/MDM.cm + pSB6A1/p5/p10/p20 -- 0mM & 0.01 mM IPTG
- Test evaluation GFP with EZrich-medium:
-> Wild type
-> DH5a + pTGD-ccd-Pmb1GFP-CmFRT
-> Strains with GFP: 8/MDM.cm + pSB6A1/p5/p10/p20 -- 0mM/0.01mM/0.05mM & 0.2mMM IPTG
- Experiment 6
- Restriction of CcdB operon and pSB1C3 with EcoRI and SpeI
-> Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
Restriction Digest T7-ccdB: 14.4 ng/µl
Restriction Digest pSB1C3: 4.4 ng/µl
- Ligation of CcdB operon and pSB1C3: 25 minutes at room temperature
- Transformation in E. coli DH5a, using heat shock (42°C),
-> Plate on chloramphenicol plate and grow overnight at 37°C : negative
- Q5 PCR of pSB1C3, using the designed Gibson primers and the linearised plasmid
-> Purification of PCR-product, using Qiagen Qiaquick PCR purification kit: nanodrop:
pSB1C3-backbone: 109.9 ng/µl
- Gibson Assembly (1h at 50°C)
- Transformation in E. coli Top10 subcloning cells, using electroporation (Time Constant: 4.4)
-> Plate on chloramphenicol plate and grow overnight at 37°C: positive
- Colony PCR (Crimson) using primers MDM0606 and MDM0607:
6 colonies can possible be positive (but it is not sure)
-> Inoculate the 6 colonies (overnight)
- Control of fragment of pSB1C3 on a gel => Backbone is not present
- Plasmid purification of the 6 colonies + restriction to control of they are the good colonies (on a gel)
There is a possibility that they are the good ones, so we will send it to sequenate
-> pSB1C3-T7ccdB sent for sequencing with primers MDM0606, MDM0607, MDM0060 and MDM0039: negative
- Q5 PCR of pSB1C3, using the designed Gibson primers and the linearised plasmid
-> Purification of PCR-product, using Qiagen Qiaquick PCR purification kit:
nanodrop: pSB1C3-backbone: 58.0 ng/µl
-> Control of fragment of pSB1C3 on a gel => Backbone is not present
- Q5 PCR of pSB1C3, using the designed Gibson primers and the restriction digest of pSB1C3
-> Purification of PCR-product, using Qiagen Qiaquick PCR purification kit:
nanodrop: pSB1C3-backbone: 76.2 ng/µl
-> Control of fragment of pSB1C3 on a gel => Backbone is present
- Gibson Assembly (1h at 50°C)
- Transformation in E. coli Top10 subcloning cells, using electroporation (Time Constant: 4.3)
-> Plate on chloramphenicol plate and grow overnight at 37°C: positive => many colonies
- Colony PCR (Crimson):
We tested 48 colonies (using primers MDM0606 and MDM0607) and 4 colonies show the right fragment (2520 bp)
-> Inoculate these 4 good colonies
- Plasmid purification of the 4 colonies, using Qiagen Qiaprep Spin minikit: nanodrop:
-> pSB1C3 strain 1: 94.2 ng/µl
-> pSB1C3 strain 2: 163.4 ng/µl
-> pSB1C3 strain 3: 160.2 ng/µl
-> pSB1C3 strain 4: 124.3 ng/µl
- Restriction with BspHI to control on a gel of they are the right colonies
(expected fragments are 1028bp and 3248 bp): positive
-> Strains 2, 3 and 4 seems to be the correct colonies
- pSB1C3-T7ccdB of these 3 colonies sent for sequencing with primers MDM0606, MDM0607, MDM0060 and MDM0039
-> sequence shows a mutation in the stop-codon, so we have to start over again
Close
Week 2: september 9 - 15
- Experiment 2
- Q5 PCR for amplification of T7ccdB-fragment, using the designed Gibson primers, on p5SpFRT-T7ccdB, p10SpFRT-T7ccdB and p20SpFRT-T7ccdB
-> Using DpnI at 37°C for 1 hour
-> Purification of PCR-product by using minElute reaction cleanup kit
-> Control on gel: negative
- Gibson Assembly (1h at 50°C) of pSB3T5 and CcdB operon that we already amplified before
- Transformation in E. coli Top10 subcloning cells, using electroporation (Time Constant: 3.0)
-> Plate on tetracyclin plate and grow overnight at 37°C: negative, there are oly red colonies
- Gibson Assembly (1h at 50°C) of pSB4A5 and CcdB operon that we already amplified before
- Transformation in E. coli Top10 subcloning cells, using electroporation (Time Constant: 4.0)
-> Plate on ampicillin plate and grow overnight at 37°C: positive
- Colony PCR (Crimson): there is only 1 colony.
We tested this colonie (using primers MDM0606 and MDM0607) and it show the right fragment (2520 bp) on gel
-> Inoculate these colonie
- Plasmid purification of the colonie, using Qiagen Qiaprep Spin minikit: nanodrop:
- Experiment 4
- Experiment 6
- Q5 PCR for amplification of T7ccdB-fragment, using the designed Gibson primers, on p5SpFRT-T7ccdB, p10SpFRT-T7ccdB and p20SpFRT-T7ccdB
-> Using DpnI at 37°C for 1 hour
-> Purification of PCR-product by using minElute reaction cleanup kit
-> Control on gel: negative
- Gibson Assembly (1h at 50°C), using a T7ccdB we already amplified before
- Transformation in E. coli Top10 subcloning cells, using electroporation (Time Constant: 4.0)
-> Plate on chloramphenicol plate and grow overnight at 37°C: negative
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