Team:Heidelberg/Notebook/Killing II/5thweek

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Latest revision as of 21:27, 28 October 2008

5^th week

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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.
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:
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.
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

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
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

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