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Date:

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Booking


Please book the equipment listed below via our online booking system PPMS or by scanning the QR code on the system itself. You don't have a PPMS account yet? Feel free to contact us. 


Outline


  • general approach to test protein expression level under various conditions simultaneously
  • conditions to vary may be growth and expression temperature, concentration of the inducing reagent
  • the choice of growth media and expression length (e.g. 4h or over night)
  • a variety of E. coli host strains and constructs can be tested on a short time frame
  • results of the first screening can be a starting point for further up-scale experiments
  • E. coli culture expression samples are taken before induction, each 1h after induction and before harvesting
  • samples are diltued to the same OD600 for easier comparison
  • expression is analyzed via SDS PAGE and Western Blot 
  • in addition, protein solubility can be tested on the fly



To Do


please ensure the following is at hand and prepared in sufficient amounts

  • Incubator Innova S441
  • XCell SureLock SDS PAGE system
  • BioRad TransBlot Turbo
  • fresh streak transformation with your plasmid and E. coli hosts of interest:

  • LB medium for preculture
  • 1x 100mL growth medium of choice (LB, TB, Auto Induction) in 250mL flask per condition to be tested
  • selection marker:

  • inducing reagent (IPTG, AHT, arabinose):

  • SDS PAGE with the desired concentration
  • Western Blot reagents



Day 1, date:


  • Overnight Culture
    • mix 6mL LB with 6µL of your desired selection marker in a 15mL falcon tube
    • pick single colony from fresh streak transformation on LB-Agar plate
    • transfer into 6mL LB and close the falcon tube
    • growth at ______ °C, ______ RPM over night (e.g. 37°C, 180 RPM)


Day 2, date:


  • Testexpression Culture
    • add 100µL desired selection marker to 100mL growth medium of choice in 250mL flask
    • inoculate with ______ mL overnight culture (e.g. 2-5mL)
    • measure starting OD600
    • follow cell growth at ______ °C, ______ RPM (e.g. 37°C, 180 RPM) measuring OD600 until
      • LB media: OD600 = 0.6
      • Auto Inducing Media: OD600 = 0.6
      • TB media: OD600 = 1.2


Sample





timeOD600OD600OD600OD600OD600OD600

















































t0





table 01 - time / OD600 until t0

  • take 1mL sample for SDS PAGE/Western Blot before induction (t0)
    • spin down 1mL sample in 1.5mL Eppi at fullspeed in tabletop centrifuge
    • discard supernatantm make sure no liquid remains in the Eppi
    • freeze pellet in liquid nitrogen, annote OD600 to every frozen sample
    • keep at -20°C until further use
  • Induction
    • LB/TB media: induce expression with ______ mM inducing reagent (IPTG, AHT, arabinose) (e.g. 0.1mM - 1mM)
    • Auto Inducing Media: switch temperature from ______ °C (e.g. 37°C) to ______ °C (e.g. 20°C or 30°C)
  • measure OD600 each 1h after induction
  • take 1mL samples each 1h, up to 5h after induction (t1, t2, etc)
    • proceed with 1mL samples as described above
  • annotate OD600 to specific timepoints in table 02


Sample





timeOD600OD600OD600OD600OD600OD600
t1





t2





t3





t4





t5





table 02 - measuring points after induction with different temperatures

  • eventually keep cultures growing overnigh



Day 3, date:


  • Test Expression Culture and SDS PAGE and Western Blot
    • before ending the expression experiment, measure OD600 and take another 1mL sample for SDS PAGE and Western Blot analysis
      • proceed with 1mL samples as described above
Sample





timeOD600OD600OD600OD600OD600OD600
t over night





table 03 - OD600 after overnight growth

  • depending on the number of samples you need 1-2 SDS PAGE for analysis
    • 1x SDS PAGE
    • 1x Western Blot
  • Loading the Gel
    • please ensure to only take one sample from -20°C at a time, to avoid a sticky mess while
    • prepare 5µL of 4x SDS Loading Dye / Sample in 1.5mL eppi
    • take out sample from -20°C, briefly warm up in hands
    • dilute sample with ddH2O
      • 100µL ddH2O/OD600=0.1
      • e.g. OD600=0.6, dilute pellet with 600µL ddH2O
    • immediately transfer 20µL of resuspended cell pellet to 5µL 4x SDS Loading Dye and mix well
    • heat sample to __________ °C for __________ min. (optional)
    • load __________ µL of prepared sample into SDS PAGE gel (depending on the wellcomb used, 7-10µL)
      • if you are using two gels, please ensure to load both gels in the same order for better compairson of SDS PAGE and Western Blot
    • continue with all remaining samples as described above
    • use PreStained Marker for Western Blot
  • annotate your sample sequence here









  • run SDS PAGE and Western Blot referring to our standard protocols (SDS PAGE: constant 120V, 90min. Western Blot: 25V, 1.0A for 30min (BioRad TransBlot Turbo))


Results and Conclusions