Team:Heidelberg/Notebook/Killing II/12thweek

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12th week

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Contents

Monday 10/20/2008

pSB1A3-Receiver-Colicin cloning

  • Minipreps of colE1/E9/E9lys-Receiver with QiaCube, Qiagen
  • Send probes to GATC for sequencing


Sender cloning: constitutive promotor-sender

  • Minipreps of J23107-Receiver with QiaCube, Qiagen
  • Send probe to GATC for sequencing


Tuesday 10/21/2008

Sequencing results of ready cloned BioBrick parts

EcoRI-site mutation PstI-site 1 mutation PstI-site 2 mutation PstI-site 3 mutation Prefix Suffix complete sequence
colE1_BB_57-1 + + + + + + +
colE1_BB_57-2 + + + + + + +
colE1_BB_57+2 ? + + ? + ? missing sequence
colE1_BB_67+1 + + + + + ? sequencing failure
colE9_BB_(2) + XXX XXX XXX + + +
colE9lys_BB XXX XXX XXX XXX + + +
sender_BB XXX XXX XXX XXX + + +

--> colE1_BB_57-1, colE9_BB_(2), colE9lys_BB and sender_BB gave positive sequencing results in all criteria and will be sent to the registry

Controldigestion of parts with EcoRI and PstI

  • colE1_BB_57-1, colE9_BB_(2), colE9lys_BB and sender_BB was digested with EcoRI and PstI: 1h 30 min -> 37 °C
 0.5 µl EcoRI (NEB)
 0.5 µl PstI (NEB)
 3.0 µl DNA
 2.0 µl EcoRI Buffer (NEB)
 2.0 µl BSA 10x
12.0 µl H2O
-------
20.0 µl
  • Gelresults: The digestion pattern looks like expected. 1% Agarose, 135 V, 30 min
081021-controldigestion.jpg

Control of antibiotics resistance

  • LB-media containing tetracycline, chloramphenicol, ampicilin or kanamycin was inoculated with the different parts to the theri antibiotics resistance.

Wednesday 10/22/2008

  • Antibiotics test: Each part only grew in ampicilin media as expected

Thursday 10/23/2008

pSB1A2-Receiver-Colicin cloning

HisTag cloning of Colicins for purification

Colicin activity test

Friday 10/24/2008

Sender activity test

Constitutive sender and amplifier ([http://partsregistry.org/Part:BBa_I15030 BBa_I15030]) activity test

  • over the day:
    • 7x Inoculation of 8 ml TB media with 160 µl from sender ONC
    • 7x Inoculation of 8 ml TB media with 160 µl from amplifier ONC
    • Inoculation of 7 ml TB media with 7 ml from GFP-receiver ONC
    • every hour (starting at t = 0 h until t = 7 h)
      • Measurement of optical density (OD) of ONC dilutions
      • Two different adequate dilutions of the diluted ONC were plate on LB Agar plates for cfu determination
      • Creation of supernatant of the measured probe by sterile filtration (storage of the supernatant at 4 °C)
  • evening: Measurement of the amount of produced AHL in the supernatants of the different timepoints
    • each: 400 µl of the respective supernatant + 400 µl fresh TB media + 200 µl T9002 cells
    • reference: T9002 cells + different concentrations of AHL
    • plate scheme:
      081011-plate scheme sender amplifier test.jpg

Saturday 10/25/2008

Results of the Sender activity test

081024 sender amplifier OD LZZ.jpg





Characterization: ColicinE1-Receiver Activitytest: Killer-prey system test and lysis test of killer cells

  • afternoon: Inoculation of the following cultures:
    • constitutive Sender with GFP(J23107-F1610 + I20260)(TB-Kana_Amp)
    • ColE1Rec pBAD-mCherry (TB-Kana-Amp-Arab)
    • T9002 without GFP pBAD-mCherry (TB-Kana-Amp-Arab)
    • ColE1Rec + I20260 (TB-Kana-Amp)
    • T9002 without GFP + I20260 (TB-Kana-Amp)
  • 8.30 pm: Preparing mixtures for the plate:
    • for colE1-Receiver killer-prey test (left part of the plate):
    • 1:4 100 µl Sender + 400 µl Receivercells
    • 1:1 100 µl Sender + 100 µl Receivercells + 300 µl TB media
    • 5:1 100 µl Sender + 20 µl Receivercells + 380 µl TB media
    • 10:1 100 µl Sender + 10 µl Receivercells + 390 µl TB media
    • 25:1 100 µl Sender + 4 µl Receivercells + 396 µl TB media
    • 50:1 100 µl Sender + 2 µl Receivercells + 398 µl TB media
    • 100:1 100 µl Sender + 0.4 µl Receivercells + 400 µl TB media
    • for colE1-Receiver lysis test (right part of the plate)
    • colE1 + I20260: 250 µl colE1 cells + 250 µl TB-Amp-Kana
    • T9002 without GFP + I20260: 250 µl T9002 without GFP + I20260 cells + 250 µl TB-Amp-Kana
  • plate scheme:
081025 killer prey lysis.jpg

Theoretical work for documentation

Sunday 10/26/2008

Theoretical work for documentation.

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