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Name:

Date:

Project:



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 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
  • 25mL growth medium of choice (LB, TB, Auto Induction)
  • Self-adhesive metal disk (for lid lifting)
  • selection marker:

  • 4mL, 20mM IPTG in 15mL falcon tube
  • SDS PAGE with the desired concentration
  • Western Blot reagents
  • TECAN Spark
  • SBS 24well micro plate: Sarstedt TC-Platte 24 Well,Standard,F; 83.3922.005)

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, set up for 24well plate (SBS 24well micro plate: Sarstedt TC-Platte 24 Well,Standard,F; 83.3922.005)
  • preset the TECAN Spark to 37°C
  • start the cooling device and set to the temperature the TECAN should reach after induction
  • add 1µL of selection marker to each well if needed
  • add 1mL of medium to each well, if needed
  • inoculate with 0.01 mL overnight culture (see scheme below in table 01, positions A1 to A6 are fixed)
  • Put some double sided tape onto a magnet, and place it in the middle of the lid
  • Put the lid onto the plate


  • injector preparation
    • open the Injector box and transfer the carbon needles from both channels to a beaker filled with milliQ
    • Rinse 5 times (1500 µL, standard setting) both channels with water
    • transfer needle A (left) to a falcon tube with IPTG, put both into a falcon tube stand
    • Prime channel A with 20 mM IPTG (1500 µL, standard)
    • Put the injector rod into the SPARK (till it ‘clicks’)


  • Put the 24well plate into the preheated SPARK plate reader
    • SDS plate is incubated by shaking in orbital mode with 5mm leverage at 120 RPM
    • every 15min (=1 cycle), the lid is liftetd and the absorption at 600nm is measured of every well (OD600)
    • when the control wells (reference) are rising higher as OD600 = 0.2 (corresponds to OD600 = 0.5 in a 1cm cuvette) two events are triggered



  • Load the SOP method according to the experiment you want to run
    • here:
A1blankA2referenceA3blankA4referenceA5blankA6reference
B1
B2
B3
B4
B5
B6
C1
C2
C3
C4
C5
C6
D1
D2
D3
D4
D5
D6

table 01 - experimental setup

  • Induction with IPTG
    • the volume of 20mM IPTG to be added to the wells can be set in the method editings




Day 3, date:


  • Test Expression Culture and SDS PAGE and Western Blot
    • check the latest OD600 in the TECAN software and dilute each well of interest in an Eppi to a 20µL sample with an OD600= 0.1


  • Put out your plate and other consumables
  • Remove the injector rod from the SPARK and put it in the holder left to the injector
    • Backfill your injected solution (discard or keep the solution as needed)
    • Rinse (wash) the used injector lines with water at least 5 times
    • Rinse the lines with air in order to dry the tubing
    • Empty and clean the trash container below the injector rod
    • Put the injector rod back into the Holder with detergend (1% Extran)


  • depending on the number of samples you need 1-2 SDS PAGE for analysis
    • 1x SDS PAGE
    • 1x Western Blot
  • Loading the Gel
    • add 5µL of 4x SDS Loading Dye / Sample in 1.5mL eppi
    • 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
    • use PreStained Marker for Western Blot
  • run SDS PAGE and Western Blot referring to our standard protocols (SDS PAGE: constant 150V, 80min. Western Blot: 25V, 1.0A for 30min (BioRad TransBlot Turbo))


Results and Conclusions