Team:ETH Zurich/Wet Lab/Protocoll

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Introduction of GFP/RFP into MG1655/MD42 E. coli strains

using the Lambda Red System

 

Subcloning of GFP/RFP into pCK01 followed by PCR amplification

 

Day 1: Friday, July 11

  • digest p18GFP/BB_J14461RFP plasmids and pCK01 2.5 h

    • 25 µL GFP construct/RFP construct/pCK01 plasmid

1 µL EcoRI

1 µL PstI

3 µL EcoRI buffer

 

→ 2 h at 37°C

→ pCK01 digest was heat-inactivated for 20 min at 80°C

 

  • run gel to confirm digest 1 h

    • 0.8 % agarose gel:

lane 1 (large!) + 2: 26 µL RFP digest + 6 µL loading buffer (LB)

lane 3: 4 µL RFP digest + 2 µL LB

lane 4: 2.5 µL λ marker + 2 µL LB

lane 5 (large!): 26 µL GFP digest + 6 µL LB

lane 6: 4 µL GFP digest + 2 µL LB

 

→ run at 90 V for ca. 1 h

lanes 3/4 and lane 6 cut out and put in EtBr for ca. 30 min

 

pics (4 s exposure) saved under GFP_RFP_restr

 

  • cut out GFP/RFP bands, recover DNA 1 h

    • RFP showed 2 bands: one at ca. 1000 bp and one higher

(ca. 1000 bp + higher expected)

    • GFP showed 3 bands: one at ca. 1000 bp, one higher and one lower

(ca. 500, 1000 and 2200 bp expected)

 

→ bands at 1000 bp were cut out (ca. 250 mg)

 

    • gel extraction was performed according to protocol:

750 µL of buffer QG

250 µL isopropanol

elution with 30 µL EB buffer (wait 1 min before elution!)

 

  • ligate recovered DNA with digested pCK01 O/N

    • 3 µL pCK01 vector

1 µL T4 buffer

0.5 µL T4

5.5 µL GFP insert/RFP insert/ water (control)

 

                        → spin down and put to 4°C O/N

 

Day 2: Saturday, July 12 (5 pm)

  • transform ligations (3 x) and GFP/RFP/pCK01 plasmids 2 h

(to get backups): 6 transformations!

    • 3 ligations transformed:

10 µL ligation (GFP/RFP/control)

40 µL competent cells

    • 3 backup plasmids transformed:

2 µL p18GFP/BB_J14461 RFP/pCK01 plasmids

40 µL competent cells

 

→ 20 min on ice

→ 90 sec at 42°C

→ 90 sec on ice

→ 200 µL CVM added

→ 1 h recovery at 37°C and 220 rpm

 

  • plate out transformations (6 x) and incubate at 37°C O/N

    • 3 ligations (GFP-pCK01, RFP-pCK01, control) plated on

LB + chloramphenicol (cat) + x-gal agar plates

    • pCK01 plated on LB + chloramphenicol agar plates, p18GFP and BB_J14461 RFP plated on LB + ampicillin agar plates

 

→ put to 37°C O/N

 

Day 3: Sunday, July 13 (5 pm)

  • pick colonies and inoculate over night cultures: O/N

1 colony of GFP/RFP/pCK01 and lambda red

+ 2 x 5 of GFP-pCK01/RFP-pCK01 → 14 cultures

    • one GFP/RFP/lambda red colony was inoculated in 15 mL-Falcon

tubes containing 5 mL LB medium + ampicillin

    • 5 colonies of GFP-pCK01/RFP-pCK01 were inoculated in 5 mL

LB medium + chloramphenicol

 

→ put to 37°C O/N

 

Day 4: Monday, July 14 (9 am)

  • do mini preps of GFP-pCK01, RFP-pCK01 (3 x each) and GFP/RFP/pCK01/lambda red plasmids (10 mini preps):

    • eluted in 50 µL EB buffer after 1 min incubation at RT

 

  • digest and gel to check cloning:
    • GFP/RFP plasmids digested:

                    2 µL DNA

                    1 µL EcoRI buffer

                    0.5 µL EcoRI

                    0.5 µL PstI

                    6 µL water

 

    • GFP-pCK01/RFP-pCK01/pCK01 plasmids digested:

                    8 µL DNA

                    1 µL EcoRI buffer

                    0.5 µL EcoRI

                    0.5 µL PstI

 

                    → put to 37°C for 2 h

 

    • run on 0.8 % gel:

                    add 2 µL LB

                    load 10 µL of each

                    run at 100 V

                    take pictures

 

                    → hardly any DNA visible

 

  • Inoculation of an additional 3 colonies of GFP-pCK01 and RFP-pCK01
    • inoculated in 5 mL chloramphenicol containing LB medium
    • incubated at 37°C O/N

 

Day 5: Tuesday, July 15

  • GFP-pCK01, RFP-pCK01 (3 x each) and GFP/RFP/pCK01/lambda red plasmids were run on 0.8% gel without digestion

          → double bands (several colonies picked?!)

  • Mini prep of 2 x 3 GFP-pCK01/RFP-pCK01 cultures
    • mini preps (2 x 2) were performed and run on 0.8% gel

                    → 3/4 positive

 

Day 6: Wednesday, July 16

  • do PCR of GFP-pCK01 and RFP-pCK01 constructs using the ordered primers
    • primers were dissolved in xxx µL/xxx µL ddH2O (forward/reverse)
    • Master-Mix (MM):

                    40 µL 5 x Taq buffer

                    32 µL 25 mM MgCl2

                    2 µL ATP

                    2 µL GTP

                    2 µL CTP

                    2 µL TTP    

                    8 µL forward primer (100 µM)

                    8 µL reverse primer (100 µM)

                    8 µL Taq polymerase

                    272 µL ddH2O

    • 3 different amounts of GFP-pCK01 (no. 2, 15.07.08)/RFP-pCK01 (no. 1, 15.07.08) template were used:

                    49 µL MM + 1 µL template

                    48 µL MM + 2 µL template

                    46 µL MM + 4 µL template

 

                    → 6 PCRs

 

    • PCR program:

                    5 min 95°C

 

                    35 x

                    1:50 min 95°C

                    1:50 min 50°C

                    2 min 72°C

                    

                    5 min 72°C

                    4°C after finish; running time: ca. 4:30 h

 

  • check and purify PCR product on gel
    • 10 µL LB added to all 6 PCR products
    • 6 µL loaded for checking
    • rest (ca. 50 µL) loaded in large pockets for extraction
    • 6 µL lambda marker loaded (3 µL marker + 3 µL LB)
    • run at 100 V on 0.8% gel

            

                → no bands, PCR needs to be repeated!
 
Day 7: Thursday, July 17
  • repitition of PCR with changed protocol
    •  2.5 µL high fidelity buffer (with MgCl2, no.2)
                     1 µL primer up (10 µM!)
                     1 µL primer down (10 µM!)
                     0.5 µL dNTPs (10 µM of each dATP, dGTP, dCTP, dTTP; is aliquoted)
                     0.5/1 µL template (GFP-cat 15.07.08 no. 3, RFP-cat 15.07.08 no. 1)
                     0.5 µL high fidelity polymerase 
                     19 µL ddH2O

 

                     → 4 PCRs a 25 µL
 
    • program:
                    5 min 94°C
                    
                    1 min 95°C
                    1 min 55°C
                    2 min 72°C
                    → 25 x
 
                    10 min 72°C
                    4°C till end
 
  • 0.8% gel to check and extract PCR product
    • 5 µL LB was added to each
    • run at 100 V
 
                    → bands at 2500 kb for all 4 samples
 
  • gel extraction
    • gel extraction was performed using 1100 µL buffer QG and 350 µL isopropanol                 
    • samples were eluted with 30 µL EB buffer (wait 1 min before centrifugation!)

 

Day 8: Friday, July 18
  • run extracted PCR products on 0.8 % gel to check extraction and size
    • Agarose gel electrophoresis of PCR products (2µl 100 bp Fermentas DNA Marker; 3µl PCR product GFP 0.5, GFP 1, RFP 0.5, RFP 1)
    • PCR product is larger than 1kb, yield exceeds 30ng/ul
 
                    → clear bands for all 4 PCR products!
 
<img alt="" id="khah" style="width: 389px; height: 211px;" src="http://docs.google.com/File?id=ddsp7zr4_1fkdvbchq_b" />
ready for transformation into lambda red-transformed, competent MG1655/MD42 strains (see below)

 

 

 
Preparation of competent MG1655 and MD42 strains for introduction of GFP/RFP constructs

 

Day 1: Thursday, July 10

plate out glycerol stocks of MG1655 and MD42 done

 

  • scrape off some of icy glycerol stock and plate on LB agar

 

→ incubate at 37°C O/N

 

Day 2: Friday, July 11

  • put colonies to 4°C

    • colonies have grown and are ready to be picked!

 

Day 3: Monday, July 14

  • prepare competent MG1655/MDS42 cells

    •  2 colonies of MG1655 and MDS42 each inoculated in 5 mL LB medium (5 pm)

    • incubated at 37°C O/N
       

Day 4: Tuesday, July 15

  • prepare competent MG1655/MDS42 cells (continued)
    • 50 mL LB medium were inoculated with 500 µL over night cultures and grown to OD(600) 0.488 (MG1655) and 0.503 (MDS42)

                    (MG1655 grew faster and was stored on ice while MDS42 was still growing)

    • cultures were transfered to 50 mL Falcon tube (on ice!) and spun down for 10 min at 2500 rpm and 4°C
    • discard supernatant
    • pellet was resuspended in 50 mL buffer 1 (pre-cooled!)
    • store on ice for 90 min
    • spin 10 min at 2500 rpm and 4°C
    • discard supernatant
    • resuspend pellet in 2 mL ice cold buffer 2
    • 200 µL each were filled in 1.5 mL Eppis and stored at -80°C

 

  • transform competent MG1655/MDS42 cells with lambda red constructs (pKD46)
    • 2 µL pKD46 were transformed into 50 µL of competent MG1655/MDS42 according to the protocol
    • heat shock at 42°C
    • 200 µL CVM added
    • recovery at 30°C
    • plated on ampicillin containing agar plates at 30°C (!) O/N

 

 Day 5: Wednesday, July 16

  • prepare competent lambda red MG1655/MD42 pKD46 cells

    • inoculate 2 colonies of each MG1655/MDS42 pKD46 in 5 mL ampicillin containing LB medium

    • incubate at 30°C O/N at 200 rpm

 

Day 6: Thursday, July 17

  • prepare competent lambda red MG1655/MD42 pKD46 cells (continued)
    • protocol as described on day 4 (with MG1655/MD42 pKD46 cells!)
    • grown to OD(600) of 0.458 (MG1655)/0.536 (MDS42)
                    Again, MG1655 grew faster!
    • cells were stored on ice for 20 min before 1st centrifugation
    • aliquoted in 2 x 11 Eppis a 200 µL and stored at -80°C

 

Day 7: Friday, July 18

 

  • transform MG1655/MD42 pKD46 cells with GFP-pCK01/RFP-pCK01 PCR products (see above)

    • temperature shock transformation of MG1655/MD42 pKD46 cells with GFP-pCK01/RFP-pCK01 PCR products according to protocol:
                    For the transformation, 50ul of competent cells and 2ul of PCR product (amounting to approximately 100 ng) were used and streaked onto LB-Cat plates.
                    Incubation at 30°C for 12 hours to three days did not yield any colonies.
 
                    
                    → Transformation has failed and needs to be repeated.
 
Day 8: Tuesday, July 22
 
  • Repetition of PCR product transformations:
    • 1 µL/5 µL/10 µL of GFP-cat PCR product (0.5) and 2 µL pCK01 (control) were transformed in 45 µL MG1655 + pKD46 cells
    • 1 µL/5 µL/10 µL of RFP-cat PCR product (0.5) and 2 µL pCK01 (control) were transformed in 45 µL MDS42 + pKD46 cells
    • 10 min on ice, heat shock at 42°C, recovery with 200 µL CVM at 37°C, plated on chloramphenicol containing agar plates, incubation at 37°C O/N


Day 9: Wednesday, July 23

  • Picking colonies for O/N cultures of MG1655/MD42 pKD46 in 5mL LB medium with ampicillin.


Day 9: Thursday, July 24

    Production of competent cells with induction of lambda-red-recombination system:

  • Inoculation with 500 uL O/N culture of MG1655/MD42 pKD46 in 50mLB medium with ampicillin and 500 uL 1M Arabinose. \
  • growth until OD(600nm) = ~ 0.4 in 30C! shaker (MG1655 grew slightly faster and were put on ice 15 min earlier).
  • cells on ice for 30 min
  • centrifuge cells for 10 min, 2500 rpm, 4°C, discard supernatant
  • wash cells twice with 10 mL ice-cold ddH2O and twice with 10 mL 10% Glycerol.
  • cells resuspended in 500 µL 10 % glzcerol and aliquoted to 50 µL per Eppi
  • stored at -80°C
    

    Transformation with insert through electroporation:

  • add to 50 uL competent cells (MG1655/MD42 pKD46) 5 uL/10 uL insert (GFP-cat-1/RFP-cat-1 from PCR of 17.7.08)
    <tbody id="fz.:0"> </tbody>
      Strain Insert  
        Amount Which
    1 MG1655 5 uL GFP-cat
    2 MG1655 10 uL GFP-cat
    3 MD42 5 uL RFP-cat
    4 MD42 10 uL RFP-cat
  • put into ice-cold electroporation-tube
  • pulse with 1.25 kV, add immediately 1 mL LB
  • put into a sterile epi and recover in 37C shaker for 1 h
  • centrifuge 1 min, 12 000 rpm, discard supernatant, resuspend in residual medium
  • plate on Cat-plates and incubate O/N at 37C


Day 10: Friday, July 25

    Result: Transformation has failed and needs to be repeated.

Day 11: Monday, 28 July

    Repetition of transformation through electroporation incl. control

  • add to 50 ul competent cells (MG1655/MD42) from 24. 6.08 insert (1 uL pUCP26, contains tet-resistence or 3ul RFP-cat/GFP-cat)
  • electroshock at 1.25kV, add immediately 1mL LB + 10 uL 1M arabinose
  • recover in 37C shaker for 3 hours
  • plate 50% and incubate O/N at 37C
  • let 50% of cells on the bench in 1ml LB O/N

   

<tbody id="o4ny0"> </tbody>
  Strain Insert Electro shock time Plate Type
1 MG1655 pUCP26 6ms Tet
2 MD42 pUCP26 5.9ms Tet
3 MD42 RFP-cat (0.5 PCR 17.7.08) 5.8ms Cm
4 MG1655 GFP-cat (0.5 PCR 17.7.08) 5.9ms Cm


Day 12: Tuesday, 29 July

    Results from electroporation from 28.7.08


  • control strains (transformation with pUCP26) are grown: => electroporation has been successful
  • GFP/RFP-cat insertion failed again => integration of insert into chromosome has failed


    Test of Insertion of GFP/RFP into Target gene TnaA

  • one colony found on plate from 24.7.08, MD42 transformed with 10uL RFP-cat 
  • inoculation of colony in 5 ml LB with Chloramphenicol, and incubation in 37C shaker (start 11am - about 3/4pm until OD = 2)
  • Tryptophan-Color-Test: positive ! addition of Kovac's Regent for indoles stayed yellow, and didnt turn red. Therefore TnaA is inactive, probably by insertion of RFP-cat.
  • Fluorescence microscopy: clearly moving particles were visible under LM, some red dots under FM


  • Inoculation of O/N culture in 5 ml LB with Chloramphenicol. 
  • Incubation of Backup plates from the one colony found in 37C oven.


    Growth experiment of MG1655/MD42 in LB

    Inoculate MG1655/MD42 in 5 ml LB 37 shaker over night.       


Day 13: Wednesday, 30 July

    Test of Insertion of GFP/RFP into Target gene TnaA

  • Indol-test with O/N culture from 29.7.08 OD = 4.605 again positiv: Medium stayed yellow upon addition of Kovac's Regent for indoles.
  • to do: FL after 2h in freezer
  • to do: Primer design for PCR to verify insertion


    Growth experiment of MG1655/MD42 in LB

  • Inoculation of 100ml media with O/N culture to OD(600) = 0.05: 2.358 mL MG1655 (preculture with OD(600) = 2.12) and 1.976 mL MD42 (preculture with OD(600) = 2.53)
  • OD measurements:
    
<img width="450" height="320" alt="" id="mqen" src="http://docs.google.com/File?id=dhf5kqn7_5cx2xg4d6_b" />
<img alt="" id="e_n9" style="width: 450px; height: 320px;" src="http://docs.google.com/File?id=dhf5kqn7_8cm2vzghd_b" />
  • => max growth rate:
    • MG1655: u = 1.57 1/h
    • MDS42: u = 1.10 1/h
  • => max OD600
    • MG1655: 4.55
    • MDS42: 4.55

    Digestion of Insert and pKD46

  • digestion of 8.5 uL pKD46(MG)/pKD46(MD) in 1 uL EcoR I Buffer and 0.5 uL EcoR I
  • digestion of 3 uL RFP-cat/GFP-cat in 5.5 uL ddH2O and 1 uL Buffer 0 and 0.5 uL Pst I
  • Digestion for 2h in 37°Oven
  • load with 2 uL LB, from left to right: lambda marker, pkD42 MG, pKD42 MD, GFP MG, RFP MD.
<img id="a6lq0" alt="" /><img id="a6lq1" alt="" /> <img width="137" height="333" alt="" id="w4fx" style="margin: 1em 0pt 0pt 1em; float: left;" src="http://docs.google.com/File?id=dhf5kqn7_3ff2dk4ct_b" />
<img width="138" height="332" alt="" id="i5cz" src="http://docs.google.com/File?id=dhf5kqn7_4c5wm22fb_b" />
 
Appendix
 
<img border="0" id="jgfp3" title="GeneRuler™ & O'GeneRuler™ 100 bp DNA Ladders" style="width: 300px; height: 434px;" alt="" src="generulersm0241.jpg" />

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