Team:Heidelberg/Notebook/Killing II/1stweek

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  - incubate overnight at 37 °C
  - incubate overnight at 37 °C
*Planings for Receiver - Colicin cloning:
*Planings for Receiver - Colicin cloning:
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** use part BBa_T9002 and substitute GFP with colicin genes <br> [Image: BBa_T9002.png| 400 px| center]
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** use part BBa_T9002 and substitute GFP with colicin genes <br> [[Image: BBa_T9002.png| 400 px| center]]
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** use part BBa_F2621 or BBa_F2622 insert colicin cassette behind Lux P<sub>R</sub> (MCS) <br> [Image: BBa_F2621.png| 400 px| center]  <br> [Image: BBa_F2622.png| 400 px| center]
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** use part BBa_F2621 or BBa_F2622 insert colicin cassette behind Lux P<sub>R</sub> (MCS) <br> [[Image: BBa_F2621.png| 400 px| center]]  <br> [[Image: BBa_F2622.png| 400 px| center]]
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+
===Sender part===
===Sender part===

Revision as of 20:09, 27 October 2008

1st week

go back to the overview

Contents

Monday 08/04/2008

Colicin Receiver

Cloning

  • Transformation of miniPrep DNA of Receiver+GFP part (BBa_T9002) for Glycerolstocks in TOP10 cells: 5 µl per 100 µl E. coli TOP 10 competent cells 
- start thawing 100 µl competent E. coli TOP 10 cells on crushed ice
- add 5 µl ligation
- incubate on ice for 20 minutes
- heat shock at 42 °C for 45 sec
- incubate 2 min on ice
- add 400 µl preheated LB media
- incubate at 37 °C for 1 h, 500 rpm
- Plate 200 µl on preheated LB-Amp plate
- incubate overnight at 37 °C
  • Planings for Receiver - Colicin cloning:
    • use part BBa_T9002 and substitute GFP with colicin genes
      BBa T9002.png
    • use part BBa_F2621 or BBa_F2622 insert colicin cassette behind Lux PR (MCS)
      BBa F2621.png

      BBa F2622.png

Sender part

Cloning

  • Transformation of sender (BBa_F1610) and amplifier part (BBa_I15030) (Transformationprotocol Chris): 5 µl per 100 µl E. coli TOP 10 competent cells 
- start thawing 100 µl competent E. coli TOP 10 cells on crushed ice
- add 5 µl ligation
- incubate on ice for 20 minutes
- heat shock at 42 °C for 45 sec
- incubate 2 min on ice
- add 400 µl preheated LB media
- incubate at 37 °C for 1 h, 500 rpm
- Plate 200 µl on preheated LB-Amp plate
- incubate overnight at 37 °C


Tuesday 08/05/2008

Colicin Receiver

Cloning

  • Transformation results:
    • many colonies on receiver plates
  • Transformation BBa_F2621 and BBa_F2622 in TOP 10 cells (Transformationprotocol Chris): 5 µl per 100 µl E. coli TOP 10 competent cells 
- start thawing 100 µl competent E. coli TOP 10 cells on crushed ice
- add 5 µl ligation
- incubate on ice for 20 minutes
- heat shock at 42 °C for 45 sec
- incubate 2 min on ice
- add 400 µl preheated LB media
- incubate at 37 °C for 1 h, 500 rpm
- Plate 200 µl on preheated LB-Amp plate
- incubate overnight

Activity tests

  • Inoculation of liquid cultures with TOP10 and receiver+GFP transformed TOP10 cells for a quantitative measurement of the GFP expression induced by AI-1.


Sender part

Cloning

  • Transformation results:
    • small colonies on sender and amplifier plates
  • Inoculation of liquid cultures for glycerol stocks of amplifier, sender and receiver+GFP parts: For each part we picked 6 colonies and incubated them on LB:antibiotics (1000:1) media over night


Wednesday 08/06/2008

Colicin Receiver

Cloning

  • Mail to help@registry.com to order BBa_F1610, BBa_I15030, BBa_F2621 & BBa_F2622 because several transformation attempts did not work.
  • Transformation results (08/05/2008):
    • BBa_F2621 and BBa_F2622 in TOP 10 cells were not grown
  • Glycerol Stocks of Receiver+GFP TOP10 cells (BBa_T9002)
  • PCR for amplification of BBa_T9002, BBa_F1610, BBa_I15030 
25.0 µl Phusion MasterMix
 2.5 µl Primer fw
 2.5 µl Primer rv
17.0 µl H2O
 3.0 µl DNA

program
98 °C  2 min
98 °C  30 sec        |
48 °C  30 sec        | 25 cycles
72 °C  1 min 30 sec  |
72 °C  5 min
 4 °C  constant

Activity tests

  • Quantitative measurement of GFP Expression induced by AI-1: pipetting scheme
    Quanti auftragung.png


Sender part

Cloning

  • Mail to help@registry.com to order BBa_F1610, BBa_I15030, BBa_F2621 & BBa_F2622 because several transformation attempts did not work.
  • New plan: If we do not receive the LuxI sender part from the registry, we amplify the LuxI gene from Vibrio fischeri genome. For that we have to design primers with restriction sites for pBAD MCS:
  • Liquid cultures from previous day (08/05/2008):
    • sender (BBa_F1610) transformed cells were not grown
    • amplifier (BBa_I15030) transformed cells were not grown
  • PCR for amplification of BBa_T9002, BBa_F1610, BBa_I15030 
25.0 µl Phusion MasterMix
 2.5 µl Primer fw
 2.5 µl Primer rv
17.0 µl H2O
 3.0 µl DNA

program
98 °C  2 min
98 °C  30 sec        |
48 °C  30 sec        | 25 cycles
72 °C  1 min 30 sec  |
72 °C  5 min
 4 °C  constant


Thursday 08/07/2008

Colicin Receiver

Cloning

  • Mail to help@registry.com to order BBa_F1610, BBa_I15030, BBa_F2621 & BBa_F2622 because several transformation attempts did not work.
  • Gelextraction of Receiver PCR (06 Aug 2008)

Activity tests

  • Due to the high OD and fluorescence values we use M9 media for our next AHL measurements.


Sender part

Cloning

  • Mail to help@registry.com to order BBa_F1610, BBa_I15030, BBa_F2621 & BBa_F2622 because several transformation attempts did not work.
  • New plan: If we do not receive the LuxI sender part from the registry, we amplify the LuxI gene from Vibrio fischeri genome. For that we have to design primers with restriction sites for pBAD MCS:


Friday 08/08/2008

  • Theoretical Work...


go to 2nd week

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