Team:Heidelberg/Notebook/Killing II/2ndweek

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Revision as of 15:48, 27 October 2008

2nd week

go back to 1st week

go back to the overview

Contents

Monday 08/11/2008

Colicin Receiver

Cloning

  • Inoculation of cultures Receiver BBa_F2621, Receiver BBa_F2622 and GFP-Receiver BBa_T9002 for Miniprep and Glycerolstocks (LB) as well as in M9 (tests).
  • Digestion of BBa_T9002 to control which restriction sites are available in MCS of pSB1A3: 1.5 - 2 h -> 37 °C 10 min -> 65 °C 
 5.0 µl 10x NEBuffer 3 (NEB)
 5.0 µl BSA 10x (NEB)
28.0 µl H2O
-------
50.0 µl

 2.0 µl SpeI (NEB)
 4.0 µl BglI (Fermentas
10.0 µl Miniprep DNA
 5.0 µl 10x NEBuffer 2
 5.0 µl BSA 10x (NEB)
26.0 µl H2O
-------
50.0 µl

 2.0 µl XbaI (Fermentas)
 4.0 µl BglI (Fermentas)
10.0 µl Miniprep DNA
10.0 µl Tangobuffer (Fermentas)
 5.0 µl BSA 10x (NEB)
26.0 µl H2O
-------
50.0 µl

 4.0 µl EcoRI (Fermentas)
 4.0 µl BglI (Fermentas)
10.0 µl Miniprep DNA
10.0 µl Buffer Orange (
 5.0 µl BSA 10x (NEB)
24.0 µl H2O
-------
50.0 µl

 4.0 µl PstI (Fermentas)
 4.0 µl BglI (Fermentas)
10.0 µl Miniprep DNA
 5.0 µl Buffer Orange (Fermentas)
 5.0 µl BSA 10x (NEB)
28.0 µl H2O
-------
50.0 µl
  • Transformation of BBa_F2621 and BBa_F2622 with Registry protocol



General

  • Matlab course + evaluation

Tuesday 08/12/2008

Colicin Receiver

Cloning

  • Results Receiver T9002 digestion: All restriction sites of the MCS are still available
    Digest T9002 small.png
  • Miniprep of BBa_F2622 and BBa_I2621: eluted in 30 µl H2O

Activity Test

  • Dilution of ONC 1:50
  • Activation of BBa_T9002 with AHL: 
c1 = 1 nM
c2 = 10 nM
c3 = 50 nM
c4 = 500 nM
c5 = 50 µM
  • Measurement in Tecan well plate reader hourly (7h):
    • black plate: OD & GFP
    • transparent plate: OD


Sender part

Cloning

  • Miniprep of BBa_F1610 and BBa_I15030: eluted in 30 µl H2O

Activity Test

  • Measurement of sender BBa_F1610 and amplifier BBa_I15030 activity with BBa_T9002 hourly. Incubation at 37 °C.
  • Sender/Amplifier - Receiver Ratios (constant receiver ratio): 
S/A - R
  1 - 5
  1 - 2
  1 - 1
  2 - 1
  5 - 1


Wednesday 08/13/2008

Colicin Receiver

Cloning

  • pColE9-J arrived from Kleanthous Lab University of York, UK
  • Transformation of BBa_T9002, BBa_F2621, BBa_F2622 and pColE9-J into MG1655 (Transformation protocol Chris)
  • Control digestion of parts received from iGEM HQ: BBa_ F2621 (SfcI) and BBa_F2622 (SfcI); 37 °C 
10 µl DNA
 5 µl Buffer 10x
 2 µl Enzyme
 5 µl BSA 10x
28 µl H2O
-----
50 µl
  • Results: Many fragments which cannot be differentiated.



Activity Test

  • Data evaluation with Matlab
  • Quantitative measurement of GFP expression from receiverpart T9002 induced by AI-1
    • Plot of GFP expression against time for different AI-1 concentrations
      AI-1 timeplot small.png
    • Plot of GFP expression against AI-1 concentrations for t=4h
      AI-1 t 4h small.png

Sender part

Cloning

  • Transformation of F1610 into MG1655 (Transformation protocol Chris)
  • Control digestion of parts received from iGEM HQ: BBa_F1610 (DraI) and BBa_I15030 (BsaBI); 37 °C (DraI) and 65 °C (BsaBI) 
10 µl DNA
 5 µl Buffer 10x
 2 µl Enzyme
 5 µl BSA 10x
28 µl H2O
-----
50 µl
  • Results: Many fragments which cannot be differentiated.

Activity Test

  • Data evaluation with Matlab
  • Quantitative measurement of GFP expression from Receiverpart T9002 induced by amplifierpart I15030
    • Plot of GFP expression against time for different amplifier/receiver ratios
      080812 amplifier trans no norm timeplot small.png
    • Plot of GFP expression against amplifier/receiver ratios for different times
      080812 amplifier trans no norm concentrationplot small.png
    • Surfaceplot of GFP expression against amplifier/receiver ratios and time
      080812 ampli nonorm small.png


Thursday 08/14/2008

Colicin Receiver

Cloning

  • Inoculation of liquid cultures from transformation of wednesday: BBa_F2621, BBa_F2622, BBa_T9002 and pColE9-J in LB-media with antibiotics. Incubation at 37 °C ON.

Sender part

Cloning

  • Inoculation of liquid cultures from transformation of wednesday: BBa_F1610 and BBa_I15030 in LB-media with antibiotics.

Activity Test

  • Inoculation of BBa_T9002 and BBa_F1610 in M9-Media

General

  • Preperation for Team Meeting:
    • Presentation of colicin project of tje NTU Singapore
    • Presentation of results and future prospects


Friday 08/15/2008

Colicin Receiver

Cloning

  • Glycerolstocks of BBa_F2621, BBa_F2622, BBa_T9002 and pColE9-J cultures. 
1 ml culture + 150 µl 80% Glycerol
vortex briefly
30 min Incubation at RT
-> -80 °C freezer
  • Minipreps of BBa_F2621, BBa_F2622, BBa_T9002 and pColE9-J cultures.
  • Primer Design colicinE1:
    • fwd on colicin E1 gene with BamHI site (XbaI)
    • rv on colicin E1 gene with BamHI site (SpeI)
    • rv on immunity gene with (SpeI)
    • rv on kil gene with SpeI
    • rv in front of SOS-Box (SpeI)
    • fwd immunity gene (XbaI)
    • fwd kil gene (XbaI)
  • Primer Design colicinE9:
    • fwd on colicinE9 with BamHI (XbaI)
    • rv on canonical lysis gene (SpeI)
    • rv on ColE9 (SpeI)

Activity Test

  • AI-1 measurement (see protocol Tuesdaysday for details) 8h

Sender part

Cloning

  • Glycerolstocks of BBa_F1619 & BBa_I15030 cultures. 
1 ml culture + 150 µl 80% Glycerol
vortex briefly
30 min Incubation at RT
-> -80 °C freezer
  • Minipreps of BBa_F1619 & BBa_I15030 cultures.

Activity Tests

    • Amplifier measurement (see protocol Tuesdaysday for details) 8h: but we used a 1:25 dilution of Receiver cells

General

  • Seminar on Synthetic Biology

go to 3rd week

Retrieved from "http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/2ndweek"