Team:Heidelberg/Notebook/Killing II/5thweek

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Revision as of 15:47, 27 October 2008

5th week

go back to 4th week

go back to the overview

Contents

Monday 09/01/2008

Colicin Receiver

pSB1A3-Receiver-Colicin-Cloning

  • Transformationresults of ligation pSB1A3-RecluxpR no colonies
  • Retransformation of ligation into TOP 10 -> standard protocoll

Activity Test

  • Inoculation of TOP10 ONC for colicin-test next day:
    • +2 ml MG1655
    • +0.002 µg/ml Mytomycin C
    • 2 ml ColE1 unstressed
    • 2 ml ColE1 stressed
    • 2 ml ColE9 unstressed
    • 2 ml ColE9 stressed
  • Inoculation for growthcurve measurement over the day (tomorrow):
    • ColE1 unstressed
    • ColE1 stressed
    • ColE9 unstressed
    • ColE9 stressed
    • DH5alpha
    • TOP10
    • MG1655

Sender part

pBAD-Sender Cloning

  • Double transformation of pBAD-F1610-9 and AI-2-Sender int TOP 10 and DH5 alphha
  • Inoculation of ONC with pBAD-F1610 colony 9 (from glycerolstock)

General

  • Preparation of M9 media



Tuesday 09/02/2008

Colicin Receiver

pSB1A3-Receiver-Colicin-Cloning

  • Inoculation of liquid cultures from Transformation of ligation: pSB1A3-RecluxpR.

Activity Test

Sender part

pBAD-Sender Cloning

  • Miniprep of pBAD-F1610,9: 179,6 ng/ul; Qiagen Miniprepkit
  • Inoculation of liquid cultures from Transformation of double-transformation.

Activity Test

  • Results of Colicintest: You can see that stressed Colicin E1 cells are able to kill TOP 10. But this could also be caused by Mytomycin C.
    080902-colicin-activity ON measurement small.jpg
  • Growthcurve:
    • Inoculation of TOP10s with 1ml/2ml supernatant of:
      • MG1655
      • ColicinE1 unstressed
      • ColicinE1 stressed
      • ColicinE9 unstressed
      • ColicinE9 stressed
    • OD was measured hourly in tecan plate reader
    • New inoculation of same cultures in LB media.



Wednesday 09/03/2008

Colicin Receiver

pSB1A3-Receiver-Colicin-Cloning

  • Minipreps of the ligation ONCsvfrom 02.09.: 
concentrations:
  R1: 152,3 ng/ul
  R2: 150,4 ng/ul
  R3: 76,7 ng/ul
  R4: 132,8 ng/ul
  R5: 142,1 ng/ul 
  R6: 138,4 ng/ul
  R7: 136,8 ng/ul
  R8: 187,7 ng/ul
  • Controldigestions of ligation with XbaI and SpeI 
10.0 µl DNA
 2.0 µl NEBuffer 2
 2.0 µl BamHI
 2.0 µl XbaI
 2.0 µl BSA 10x
 2.0 µl H2o
-------
20.0 µl
  • Gel of digestion:
    080903-controldigestion luxpR cloning small.jpg
  • Gelresults: Expected fragments 2150 bp and 1088 bp. The 2150 bp fragments are there but there is a 1500 bp fragment instead of the 1088 bp fragment. Despite this we made some new glycerolstocks.


Activity Test

  • Results of growth curve test from yesterday: The results were very diffusing so that we perform this test again.
    080903-growthcurve colicin test small.jpg
  • New hourly growth curve measurement (see Tuesdaysday for details). No significant effect of colicins can be seen. Only Mytomycin C kills the TOP 10 cells very effectively
    080903-growthcurve colicin test 2 small.jpg

Sender part

pBAD-Sender Cloning

  • Minipreps of doubletransformations: 
concentrations
  prey_Top10,1: 123,6 ng/ul
  prey_Top10,2: 157,4 ng/ul
  prey_DH5-a,1: 202,0 ng/ul
  prey_DH5-a,2: 174,2 ng/ul


Thursday 09/04/2008

Colicin Receiver

pSB1A3-Receiver-Colicin-Cloning

  • Despite of the strange digestion pattern we try the next step of the cloning.
  • PCR of colicins: 
25.0 µl Phusion MasterMix
 2.5 µl colE1prot/colE9prot_fw_BamHI
 2.5 µl colE1prot/colE9prot_rv_SpeI
18.0 µl H2O
 2.0 µl DNA templates
-------
50.0 µl

program:
98 °C  30 sec
98 °C  10 sec
59 °C  20 sec
72 °C  45 sec
72 °C   8 min
 4 °C  constant
  • PCR purification and analytic gel: Expected fragments are there
    080904-colicin PCR purification.jpg
  • Digestion of purified PCR products with BamHI and SpeI: 1h -> 37 °C 
Vector:
 6.0 µl DNA
 2.0 µl NEBuffer 2
 2.0 µl SpeI
 2.0 µl BamHI
 2.0 µl BSA10x
 6.0 µl H2O
-------
20.0 µl

Insert:
10.0 µl DNA
 2.0 µl NEBuffer 2
 2.0 µl SpeI
 2.0 µl BamHI
 2.0 µl BSA10x
 2.0 µl H2O
-------
20.0 µl
  • Gel & Gelextraction: The expected bands were visible. After cutting we purified them using the Qiagen Gel Extraction Kit.
  • Sequencing: Send R1 (LuxR-Receiver) to GATC.
  • Inoculation of 4 receiver colonies

Activity Test

  • New plans for characterization:
    • Cloning of Colicin proteins into pQE-30 vector for His-Tag purification of colicins. Therefore we designed reverse primers with HindIII (ColE1) and XmaI(ColE9) sites.

Sender part

pBAD-Sender Cloning

  • Inoculation of 4 cotransformations for activity tests


Friday 09/05/2008

Colicin Receiver

pSB1A3-Receiver-Colicin-Cloning

  • Ligation and Transformation of pRecluxpR with ColE1/ColE9: no results

Sender part

Activity Test

  • Activity test over the day: no results


go to 6th week

Retrieved from "http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/5thweek"