Team:Evry/Experiments/Protocols

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Protocols




Preparation of competent E. coli bacteria for heat shock

    WORK UNDER STERILE CONDITIONS


  • Launch a pre-culture of the strain in LB at 37 °C
  • Inoculate 25 mL of LB 1/50 with the pre-culture
  • Let it rise to 37 °C with stirring until DO = 0.5-0.7
  • In the spectrophotometer use LB medium as blank
  • Cool the cells 10 min on ice
  • Centrifuge the culture 6 min at 4000 rpm at 4 °C
  • Throw away the flow-through
  • Take the pellet in 1/2 volume of cold CaCl2 0.1 M
  • Cool the cells 20 min on ice
  • Centrifuge the culture 6 min at 4000 rpm at 4 °C
  • Throw away the flow-through
  • Resuspend the pellet in 1/50e volume of cold CaCl2 + 10 % glycerol
  • Aliquote 50 µL in Eppendorf 1.5 mL tubes
  • Store samples on ice for immediate use or freeze 50 µl aliquots in -80 °C

 

Transformation by heat shock (E. coli)
  • Thaw 50 µL bacteria (E. coli DH5-alpha) chemo-competent
  • Add 2 µL DNA (mix ligation)
  • Incubation 20 min on ice
  • Heat shock: 45 s at 42 °C
  • Incubation 3 min on ice
  • Add 450 µL LB
  • Incubation 1 h at 37 °C with shaking
  • Centrifugation 2 min at 7000 rpm
  • Throw away supernatant
  • Resuspension with 100 µL LB medium
  • Spread on plates LB-agar with appropriate concentration of antibiotic(s)


Preparation of competent P. putida bacteria for electroporation
  • Set a 5 mL preculture of putida KT2440 in LB
  • Incubate overnight at 30 °C with shaking
  • Inoculate 60 mL of LB with 1 mL of preculture
  • Incubate at 30 °C with stirring until OD(600nm)=0.6-0.8
  • Divide the culture into proper sized tubes
  • Harvest cells by centrifugation (4000 rpm, 6 min, 4 °C). Discard the supernatant
  • Resuspended the pellet in ice-cold 5 mL HEPES (1 mM)/10 mL culture.
  • Centrifugation 4000 rpm, 6 min, 4 °C
  • Discard the supernatant.
  • Resuspended the pellet in ice-cold 5 mL HEPES (1 mM)/10 mL of culture.
  • Centrifugation 4000 rpm, 6 min, 4 °C
  • Discard the supernatant.
  • Resuspended the pellet in 100 µL HEPES 1 mM, 10 % glycerol (20 µL glycerol 66 % per 100 µL of HEPES)/10 mL of culture
  • Store the cells at -80 °C.

Transformation by electroporation (P. putida)
  • Electroporate 50 µL of cells with 100 ng DNA at 1800V
  • Add 1 mL of LB
  • Incubate 1 h at 30 °C with shaking (for putida for other species use optimal growth temperature)
  • Plate on selective LB-agar


Colony PCR

Mix (25 µL total volume reaction):

  • 5 µL DreamTaq Green PCR Master Mix 2X (Thermo Scientific, #K1081)
  • 1.25 µL 10 µM Forward Primer
  • 25 µL 10 µM Reverse Primer
  • 10 µL Nuclease-free water (qs 25 µL)

+bacterial clone

PCR program:

  • 95 °C 5 min
  • 95 °C 30s
  • 50 °C 30s
  • 72 °C 2 min (depending on the time of the fragment to amplify)

30 repeats of the steps bloc from 2) to 4)

  • 72 °C 10 min
  • 10 °C infinite

 

Gene amplification PCR
Mix:
  • 10 µL Q5 reaction buffer 5X
  • 1 µL dNTP mix
  • 5 µL 10 µM Forward Primer
  • 5 µL 10 µM Reverse Primer
  • 1 µL DNA template
  • 5 µL Q5 DNA polymerase
  • 5 µL Nuclease-free water (qs 50 µL)

PCR program:

  • 98 °C 30s
  • 98 °C 30s
  • 55 °C 30s
  • 72 °C 1 min

35 repeats of the steps bloc from 2) to 4)

  • 72 °C 2 min
  • 10 °C infinite

 

Digestion and ligation were performed by using NEB enzymes with their corresponding protocols. For more information, refer to the official NEB website (digestion or ligation)

 

Gibson assembly

The NEB protocol was followed to performed Gibson assembly.

 

Antibiotic concentrations

 

Stock concentration

working concentration

Kanamycin

50 mg/mL

50 µg/mL

Gentamycin

10 mg/mL

10 µg/mL

Ampicillin

100 mg/mL

100 µg/mL

Spectinomycin

50 mg/mL

50 µg/mL

Chloramphenicol

50 mg/mL in ethanol

25 µg/mL

 

 

GenElute Plasmid Miniprep Kit (Sigma, PLN350-1KT) was used to perform miniprep of plasmid DNA from precultures of bacteria (E. coli).

DNA clean & concentrator-5 kit (#D4003S Zymo Research) or DNA PCR clean-up kit (NEB) were used to purified the DNA fragment or vectors after digestion.

 

DNA Gel extraction kit (#T1020G NEB) or Zymoclean Gel DNA Recovery Kit (D4001S ZymoResearch) were used to extract DNA from agarose gel after migration.