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    Cleaning factory: methotrexate (Archived)
    • Notes
    • Metadata
    • Relevant Items
    • Gibson Assembly® Protocol (E5510)
    • PCR Q5 HF
    Editing disabled on archived entry.

     
    Cleaning factory: methotrexate

    Thursday, 7/11/2019Aim: We successively induce the expression of the MTX degradation pathway and then of nuclease_gp3 . Thereafter bacteria were incubated in the presence of MTX. We monitored MTX biotransformation by HPLC analysis. MTX should be degrated as if there was no nuclease induction.For our cleaning factory, our 2 plasmids must not have the same replication origin to not lose one of them. Then, we amplify the replication origin 15A in pZA-15A31 and pZA-15A34 in order to put it in pBAD24 instead of the ColE1. For this, we use the Gibson assembly method.Primer sequences for changing the replication origin of pZA31 or 34image.pngFriday, 7/12/2019Table4Primer sequences for Gibson assembly to remove the replicative originsMonday, 7/15/2019Table5Table6Gel PCR 15.07.jpgWednesday, 7/17/2019Thursday, 7/18/2019Primer sequences for gibson assembly in order to remove the origin of pBAD24-MocloMonday, 7/22/2019Tuesday, 7/23/2019Primer sequences for gibson assembly in order to remove the origin of pBAD24-Moclo IMG_8718.JPGWednesday, 7/31/2019Primer sequences for gibson assembly in order to remove the origin of pBAD24-Moclo (2)Friday, 8/2/2019Thursday, 8/8/2019Table1Friday, 8/9/2019Table2PCR pbadmoclo et pbad A1 T7.pdfMonday, 8/12/2019Table3PCR pBAD-MoClo without Ori.pdfNanodrop resultsTuesday, 8/13/2019Friday, 8/16/2019image.pngMonday, 8/19/2019Tuesday, 8/20/2019image.pngWednesday, 8/21/2019Thursday, 8/22/2019Nanodrop Resultsimage.pngNanodrop Results 22/08Friday, 8/23/2019Tuesday, 8/27/2019Wednesday, 8/28/2019Methotrexate degradationOD600 cleaning factoryMTX - design-true.pptxCopie de MAnip1-I-GEM.xlsxMTX expérience I-GEM-29-08-2019.docx
    Thursday, 7/11/2019
    Aim: We successively induce the expression of the MTX degradation pathway and then of nuclease_gp3 . Thereafter bacteria were incubated in the presence of MTX. We monitored MTX biotransformation by HPLC analysis. MTX should be degrated as if there was no nuclease induction.
    For our cleaning factory, our 2 plasmids must not have the same replication origin to not lose one of them. Then, we amplify the replication origin 15A in pZA-15A31 and pZA-15A34 in order to put it in pBAD24 instead of the ColE1. For this, we use the Gibson assembly method.
    ●
    Correction of the replication origin of pZA-15A31 and pZA-15A34 : Marie_Natacha
    Nanodrop : (from Philippe's tubes)
    pZA-15A31 -> 0,161 ng/µL ; A260 0,0032 ; 260/280 -0,32 ; 260/280 -0,32.
    pZA-15A34 -> negative concentration.....
    ○
    PCR protocol :
    2,5 µL Ori_15A_F
    2,5 µL Ori_15A_R
    2 µL pZA-15A31 or pZA-15A34
    25 µL Master Mix 2X
    18 µL H2O mQ
    -> total 50 µL
    ○
    PCR program:
    98°C/30sec
    25 cycles : 98°C/10sec
    65°C/30sec
    72°C/30sec
    72°C/2min
    infinite 15°C
    ○
    agarose 1% gel, 100V 25min. Results :
    image.png
    Loading...
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    ○
    Heatshock plasmid transformation (Natacha)
    Let DH5alpha on ice for 30 min
    Add plasmid PZA34 and PZA31M2N (a few µL) in your bacterial solution and let it on ice for 30 min
    Heatshock at 42°C for 1 min
    Put the solution back in ice for 2 min
    Add 1 mL LB (+ Glu) and put the solution at 37°C for 1 h with shaking
    Spread out transformants on agar plates (50 µL, 100 µL and 100 µL concentrate)
    Put the plates at 37°C overnight
    Friday, 7/12/2019
    ●
    Plasmid extraction of pZA31, pZA34 and pSB1C3 : Arnaud
    Use of Macherey-Nagel kit "DNA and RNA purification" protocol page 18 "Low copy"
    3 extractions per plasmids pZA31 and pZA34 and 2 extractions for pSB1C3
    Analysis with Nanodrop :
    ●
    PCR for Gibson assembly of pBAD24A1/pBAD24_Moclo: Kim
    PCR of plasmid pBAD24 with or without the A1 gene.
    Expected size : pBAD24_A1 = 5,5 kB
    pBAD24_Moclo = 3,9 kB
    A1 = 1,6 kB
    Primers: pBAD24_F (9) and pBAD24_R (10)
    Tm = 62°C
    Follow the protocol "PCR Q5 HF"
    2 reactions per plasmid (50µL)
    PCR program:
    (30 cycles)
    98°C 30 sec
    98°C 10 sec
    62°C 3 min
    72°C 3 min
    72°C 2 min
    5 µL of each reaction were migrated on agarose 1%. No fragments were detected. The PCR needs to be done again.
    Monday, 7/15/2019
    ●
    PCR of pZA31, pZA34, pBAD24_A1 and pBAD24_Moclo : Arnaud_Abdel
    For pZA31 and pZA34, Primers : Ori 15 A_F and Ori 15 A_R
    For pBAD24_A1 and pBAD24_Moclo, Primers : pBAD24_F and pBAD24_R
    For 1 PCR , Vf = 90 µL
    ■
    2.5 µL per primers 10mM
    ■
    1 µL template
    ■
    25 µL Master Mix (Enzyme, dNTPs, Buffer)
    ■
    18 µL Water
    The 4 PCR are done simultaneously with the following program:
    I.
    98°C - 1 min
    II.
    98°C - 10 s
    III.
    62°C - 30 s
    IV.
    72°C - 165 s
    V.
    Repeat from step 2 (30 times)
    VI.
    72°C - 2 min
    VII.
    8°C - Infinity
    The products are analysed by electrophoresis
    Gel PCR 15.07.jpg
    Loading...
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    Wednesday, 7/17/2019
    Gibson cloning, substitute replication origin ColE1 with A15 : Hugues
    ○
    Extraction on gel of pBAD24-A1 (without Ori ColE1) with the kit DNA extraction Macherey-Nagel
    ○
    Digestion of template plasmids with DpnI, 0,5µL of enzyme (37°C 1h then 80°C 20min)
    ○
    PCR clean up Macherey-Nagel (elution in 30µL NE buffer)
    ○
    Nanodrop pBAD24-A1(without Ori ColE1) = 1,5 ng/µL
    Thursday, 7/18/2019
    ●
    Nanodrop and Gibson assembly of A15 (1) and A15(4) (Kim and Hugues):
    1µl DpnI 1h at 37°C. Then 20 minutes at 80°C. PCR clean-up.
    Nanodrop concentration:
    A15 (1) = 100 ng/µl
    A15 (4) = 100 ng/µl
    Gibson:
    1h with Gibson mix at 50°C. 1:1 ratio --> 4µl of plasmid + 1 µl of (1) and (4) each diluted at 1/10.
    ●
    PCR on plasmid pBAD24-MoClo in order to remove the replication origin: Hugues
    PCR protocol:
    25µL buffer Q5 2X
    2,5µL primer 9
    2,5µL primer 10
    1µL pBAD24 MoClo
    10µL solution Q
    9µL H2O
    PCR program used:
    98°C 30sec
    98°C 10sec
    62°C 20sec *5
    72°C 2min
    98°C 10sec
    72°C 20sec *25
    72°C 2min
    72°C 2min
    Monday, 7/22/2019
    ●
    PCR colony on bacteria transformed with Gibson products : Arnaud
    PCR program:
    I.
    98°C 5min
    II.
    98°C 30sec
    III.
    54°C 30sec
    IV.
    72°C 1min
    V.
    Repeat from step 2 (30times)
    VI.
    72°C 10min
    VII.
    8°C Infinity
    PCR protocol :
    Vf = 25µL
    dXTPs 5 mM 1µL
    Oligos (7 et 8) 1µL/oligo
    Matrice 1µL
    Enzyme DreamTaq 0.5µL
    Tampon DreamTaq 10X 2.5µL
    H2O 18µL
    Results : Analysis on eletrophoresis showed no success with the PCR
    ●
    Gibson : Arnaud
    Tomorrow:
    DpnI digestion
    PCR clean up
    Transformation into competent cells
    Tuesday, 7/23/2019
    PCR for Gibson assembly of pBAD24A1/pBAD24A1 without Ori : Colombe
    PCR of plasmid pBAD24A1 with or without the ori.
    Expected size : pBAD24_A1 = 5,5 kB = 5575 B
    Primers: pBAD24_F (9) and pBAD24_R (10)
    Tm = 62°C
    Follow the protocol "PCR Q5 HF"
    2 reactions per plasmid (50µL) : 4 reactions in total
    PCR program:
    (30 cycles)
    98°C 30 sec
    98°C 10 sec
    62°C 3 min _ 5 cycles
    72°C 3 min _ 25 cycles
    72°C 5 min
    RESULTS :
    wells 1 & 2 : pBAD24_A1 with ori => no results because of empty eppendorf ?
    wells 3 & 4 : pBAD24_A1 without ori from gel extraction of the previous PCR (Arnaud ?)
    IMG_8718.JPG
    Loading...
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    (5 µL of each reaction were migrated on agarose 1%.)
    Then DNA extraction from agarose gel with gel extraction's kit of NucleoSpinR Gel and PCR clean-up, macherey-nagel
    Seen NucleoSpin Macherey-Nagel protocol
    Wednesday, 7/31/2019
    ●
    PCR pBAD-A1 (without ori colE1) : Laetitia et Mahnaz
    Oligos 9 et 10
    DNA : pBAD24-A1 118ng (nd, PCR1) or 11,8ng (diluted 10x, PCR2) or 1,18ng (diluted 100x, PCR3)
    Enz Q5
    PCR program :
    98°C/30sec
    5 cycles : 98°C/10sec
    52°C/30sec
    72°C/3min
    20 cycles : 98°C/10sec
    72°C/30sec
    72°C/3min
    72°C/2min
    Results :
    Friday, 8/2/2019
    ●
    Gibson Assembly of pBAD-A1 (without origin) with origin 15A : Laetitia
    3 assemblies are done. The third one is a negative controle with only the pBAD-A1 without origin PCR fragment
    Assembly 1 :
    1.
    pBAD-A1 without origin PCR fragment 2,5 µL of PCR3 (about 300ng) (for PCR details, see July 31st)
    2.
    PCR Product origin 15A diluted 7 times 2,5µL
    3.
    Gibson assembly mix 15 µL
    Final volume = 20 µL
    Assembly 2 :
    1.
    pBAD-A1 without origin PCR fragment 2,5 µL of PCR1 (about 300ng) (for PCR details, see July 31st)
    2.
    PCR Product origin 15A diluted 7 times 2,5µL
    3.
    Gibson assembly mix 15 µL
    Final volume = 20 µL
    Assembly 3 (control) :
    1.
    pBAD-A1 without origin PCR fragment 2,5 µL of PCR1 (about 300ng) (for PCR details, see July 31st)
    2.
    H2O 2,5µL
    3.
    Gibson assembly mix 15 µL
    Final volume = 20 µL
    Then it is put at 50°C for one hour (PCR machine) and store in ice for transformation later on.
    ●
    Heatshock plasmid transformation (with Golden gate and Gibson done above) : Laetitia
    1.
    50 µL of ultra-competent cells DH5alpha E. coli +
    2 µL from Gibson assembly of pBAD-A1 (without origin) with origin 15A n°1 or
    2 µL from Gibson assembly of pBAD-A1 (without origin) with origin 15A n°2 or
    2 µL from Gibson assembly of pBAD-A1 (without origin) without origin 15A (control) n°3 or
    2 µL from Golden Gate of pBADMoclo with A1 and RBS_Rdrp or
    2 µL from Golden Gate of pBADMoclo alone (control)
    2. 30min on ice
    3. Heat shock 90s at 42°C
    4. 15min on ice and then add 1mL of LB and put tubes at 37°C for 1h
    5. Spread cells on LB+Amp+Glu and cells are incubated at 37°c for 24h
    Results :
    ●
    With Gibson products : No colony to be seen on any plate. Transformation didn't work. => Fail
    ●
    With Golden Gate : No colony for the transformants control (pBAD24_A1). Colonies available for the transformants pBAD24_A1_RBS_Rdrp on the plates. => Success
    Thursday, 8/8/2019
    ●
    PCR of pBAD24-A1, pBAD24-T7 and pBAD24-MoClo for Gibson assembly (Clara)
    Gibson assembly was designed in order to make all our plasmid origins compatible between each of them (pBAD24 and pZA31-Cpg2)
    For this PCR the master mix of Q5 was used, for more details, see the protocol "PCR Q5 HF". PCR products have been amplified everywhere except for the Ori part.
    The PCR program used was the following:
    However, we observed a smear after electrophoresis, so the PCR was done again the next day with diluted DNA.
    The photo of this gel wasn't saved.
    Friday, 8/9/2019
    ●
    PCR of pBAD24-A1, pBAD24-T7 and pBAD24-MoClo for Gibson assembly (Clara)
    Gibson assembly was designed in order to make all our plasmid origins compatible between each of them (pBAD24 and pZA31-Cpg2)
    For this PCR the master mix of Q5 was used, for more details, see the protocol "PCR Q5 HF". PCR products have been amplified everywhere except for the Ori part.
    The PCR program used was the following:
    DNAs were diluted 10 and 100 times pBAD-A1:? and pBAD-MoClo: 118ng/µl) except for pBAD24-T7 (plasmid concentration was 16ng/µl).
    Results:
    PCR pbadmoclo et pbad A1 T7.pdf
    pBAD24-MoClo wasn't amplified, maybe a pipetting error. Have to do the PCR for this plasmid again, the expected size should have been 3946 pb.
    Monday, 8/12/2019
    ●
    PCR of pBAD24-MoClo for Gibson assembly (Clara)
    Gibson assembly was designed in order to make all our plasmid origins compatible between each of them (pBAD24 and pZA31-Cpg2)
    For this PCR the master mix of Q5 was used, for more details, see the protocol "PCR Q5 HF". PCR products have been amplified everywhere except for the Ori part.
    The PCR program used was the following:
    DNA was diluted 10 and 100 times (plasmid concentration was 118ng/µl).
    Results:
    PCR pBAD-MoClo without Ori.pdf
    Again, a smear and no amplification. Then, pBAD-MoClo was sent to sequencing, might have a mutation where primers work?
    ●
    DpnI treatment on PCR products of pBAD24-A1, pBAD24-T7 without replication origins (Clara)
    1µL of DpnI was added to the PCR products and let 1 hour at 37°C. This step is to remove plasmid templates.
    ●
    PCR clean-up of PCR products of pBAD24-A1, pBAD24-T7 (Clara)
    See NucleoSpin Macherey-Nagel protocol
    ●
    Gibson assembly of PCR productions of pBAD24-A1, pBAD24-T7 without origins with Ori 15A (Clara)
    See the protocol "Gibson assembly protocol (E5510).
    2.5µL of each PCR products were added directly to 4 gibson mixes.
    The PCR product of the ori 15A was diluted 7 times and then added to only 2 gibson mixes in order to have two negative controls.
    Then, tubes where put at 50°C for one hour and then on ice. They stay at -20°C all night long before the transformation the next day.
    Tuesday, 8/13/2019
    ●
    Transformation of gibson assembly of PCR productions of pBAD24-A1, pBAD24-T7 without origins with Ori 15A (Clara)
    See the protocol "heat shock transformation"
    This transformation was done with the following plasmids:
    ○
    pBAD24-A1-Ori15A
    ○
    pBAD24-A1 without Ori
    ○
    pBAD24-T7-Ori15A
    ○
    pBAD24-T7 without Ori
    ○
    pBAD24-T7 with Ori
    ○
    pBAD24-RdRp with Ori
    These two last plasmids were used to see if our bacteria were really chemocopetents and if our Gibson assembly worked.
    Results: There are transformants for each genetic construction, even for plasmids without Ori (however there are less transformants without Ori than with Ori).
    The transformants are re-stripe on LB-glu-amp then incubate at 37°C.
    Friday, 8/16/2019
    PCR on colonies DH5alpha_pBADA1-Ori15A/ pBADT7-Ori15A to test the Gibson assembly
    Each plasmid (A1/T7 with Ori15A) is tested with oligos 5 and 8 and oligos 6 and 7.
    PCR : - Dream taq buffer 2,5µL
    ○
    dNTP (5mM) 1µL
    ○
    oligo forward 1µL
    ○
    oligo reverse 1µL
    ○
    taq pol 0,15µL
    ○
    colony (pick a single colony with a stick and dilute it into water) 1µL
    ○
    H20 qsp25µL
    98°C 30sec
    94°C 30sec
    60°C 30sec *25
    72°C 4min
    72°C 30sec
    image.png
    Loading...
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    Results : PCR on pBADA1 with oligos 5 and 8 didn't work, no band was amplified at 4470pb
    PCR on pBADT7 clone 1' with oligos 5 and 8 did work, we observe a single band which can match 3249pb, the theoric lenght of the PCR fragment.
    Re-stripe transformants DH5alpha_pBADA1-Ori15A on LB-glu0,2%-amp(100ug/mL) to test more transformants through PCR. Stock plates at room temperature over the weekend.
    Monday, 8/19/2019
    ●
    Obtention of pBADT7-ori15A
    Re-stripe of DH5alpha_pBADT7-ori15A clone 1' from friday 16/08 didn't work. It seems that no bacteria were stripe on the plate.
    From the DH5alpha + pBADT7-ori15A transformation plate (August, 13th), transformants are re-striped on LB-amp(100ug/mL) -glu (0.2%). Bacteria are placed at 37°C.
    ●
    Transformation of DH5alpha with pBADT7-ori15A or pBADA1-ori15A
    Add 2uL of Gibson reaction in 100uL DH5alpha, keep the mix on ice for 30min.
    Heatshock at 42°C for 45sec.
    Replace the mix on ice for 5min.
    Add 1mL LB and incubate at 37°C for 1hour.
    Spread bacteria of LB+glu(0,2%)+amp(100ug/mL) and incubate at 37°C.
    Tuesday, 8/20/2019
    PCR colony on transformants (DH5alpha + pBADT7-ori15A) from stripes of August, the 19th.
    PCR : - Dream taq buffer 2,5µL
    ○
    dNTP (5mM) 1µL
    ○
    oligo forward (n°5) 1µL
    ○
    oligo reverse (n°8) 1µL
    ○
    taq pol 0,15µL
    ○
    colony (pick a single colony with a stick and dilute it into water) 1µL
    ○
    H20 qsp25µL
    98°C 30sec
    94°C 30sec
    60°C 30sec *25
    72°C 4min
    72°C 10 min
    image.png
    Loading...
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    Results : clone 2, 3 and 5 amplified a fragment of 3200pb, this matches the lenght of the theorical amplicon (3249pb).
    Plasmid of each correct clone is sequenced.
    Wednesday, 8/21/2019
    ●
    Preculture : Natacha
    Preculuture of DH5alpha pBAD24_Ori15A clone 2, 3 and 5 in LB+amp (100ug/mL)+Glucose(0.2%) 15mL overnight with shaking at 37°C.
    Thursday, 8/22/2019
    ●
    Glycerol stock of DH5alpha pBAD24_Ori15A clone 2, 3 and 5 : Arnaud
    ●
    Plasmid extraction of DH5alpha pBAD24_Ori15A clone 2, 3 and 5 : Arnaud
    Use of Macherey-Nagel kit "DNA and RNA purification" protocol page 18 "Low copy"
    Analysis with Nanodrop :
    ●
    PCR : Arnaud
    A PCR with the three previous plasmids extracted (DH5alpha pBAD24_Ori15A clone 2, 3 and 5) is done with the following procedure : Vf = 50 µL
    1.
    Oligo 3 et 4 (2.5µL/oligo)
    2.
    Buffer Q5 2X (25µL)
    3.
    H2O (19µL)
    4.
    Plasmid (1µL)
    Program :
    1.
    98°C 30sec
    2.
    98°C 10sec
    3.
    61°C 30sec
    4.
    72°C 3min
    5.
    Repeat from step 2 five(5) times
    6.
    98°C 10sec
    7.
    72°C 30sec
    8.
    72°C 3min
    9.
    Repeat from step 6 twenty(20) times
    10.
    72°C 10min
    Analysis with eletrophoresis :
    From Left to right : Ladder , clone 2, clone 3,FAIL, clone 5
    Expected size : 4510 pb
    image.png
    Loading...
    Loading...
    The result show a size of around 4500 pb as expected. A PCR clean up can be done on those products.
    ●
    PCR Clean up : Arnaud
    Use of a Macherey-Nagel kit "PCR Clean up" protocole "PCR Clean up" p.18 on the previous PCR products.
    Analysis with Nanodrop
    Friday, 8/23/2019
    ●
    Heatshock plasmid transformation : Arnaud
    Transformation of KeioZ1F' with pSBC1B3_Cpg2,
    KeioZ1F' with pSBC1B3_teto,
    KeioZ1F' with pBAD_T7_Ori15A clone 2 + pSBC1B3_Cpg2,
    KeioZ1F' with pBAD_T7_Ori15A clone 3,
    KeioZ1F' with pBAD_T7_Ori15A clone 5,
    KeioZ1F' with pBAD_Moclo_Ori15A clone 2 (PCR + PCR Clean up of pBAD_T7_Ori15A clone 2),
    KeioZ1F' with pBAD_Moclo_Ori15A clone 3 (PCR + PCR Clean up of pBAD_T7_Ori15A clone 3) and
    KeioZ1F' with pBAD_Moclo_Ori15A clone 5 (PCR + PCR Clean up of pBAD_T7_Ori15A clone 5).
    The solutions are spread on LB + antiobiotics (chloramphenicol (100ug/mL) or ampiciline (100ug/mL) or both, with glucose 0.2%).
    Tuesday, 8/27/2019
    Preculture of KeioZ1 F' pSB1C3-tet, Keio Z1 F' pSB1C3-cpg2, KeioZ1 F' pSB1C3-cpg2 + pBADT7_ori15A
    Preculture in 5mL LB + glu 0.2% + amp (100ug/mL)+ cm (100ug/mL) (antibiotics if necessary)
    Wednesday, 8/28/2019
    Methotrexate degradation
    Prepare 3 cultures (pSB1C3-tet +ARA ; pSB1C3-cpg2 +ARA ; pSB1C3-cpg2 + pBADT7_ori15A +ARA) at OD600 = 0,1 in 10mL LB + IPTG 0,5mM + chloramphenicol (100µg/mL) + ampiciline (100µg/mL).
    Once the 0D600 reaches 0,4, add arabinose (0,2% final) in the culture.
    Incubate at 37°C for 30min with shaking.
    Dilute the 3 cultures in 1mL at OD600=0,2.
    Add 100µM methotrexate in each culture.
    Extract 200µL of culture + methotrexate, filter it (filter 0,2µm) and stock it at -20°C.
    5 timepoints (30min, 1hour, 2h, 3h). At 3h, take the OD.
    See below the protocol and the results
    MTX - design-true.pptx
    Copie de MAnip1-I-GEM.xlsx
    MTX expérience I-GEM-29-08-2019.docx

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