Team:Heidelberg/Notebook/Killing II/6thweek

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*Maxiprep of I15030 and T9002 for plasmid backbone. (Compact Prep Plasmid Maxi Kit (25), Qiagen)
*Maxiprep of I15030 and T9002 for plasmid backbone. (Compact Prep Plasmid Maxi Kit (25), Qiagen)
 +
[[https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/6thweek back]]
==Saturday 09/13/2008==
==Saturday 09/13/2008==

Revision as of 21:29, 28 October 2008

6th week

go back to 5th week

go back to the overview

Contents

Monday 09/08/2008

pSB1A3-Receiver-Colicin cloning

  • sequencing results of pSB1A3 - Receiver cloning -> cloning not sucessful
  • Primer sequences controlled

[back]

Tuesday 09/09/2008

pSB1A3-Receiver-Colicin cloning

  • Amplification of Colicins 
10   µl 5x Phusion HF Buffer
1.0  µl 10mM dNTPs
2.5  µl colE1/E9_prot_fw_BamHI
2.5  µl colE1_kil_prot_rv_SpeI/colE9_plasmid_rv_SpeI/colE9_lys_prot_rv_SpeI
2.0  µl Template DNA
0.5  µl Phusion DNA Polymerase
31.5 µl H2O
-------
50.0 µl
  • Amplification of Receiverpart (T9002) without GFP 
10   µl 5x Phusion HF Buffer
1.0  µl 10mM dNTPs
2.5  µl T9002_lux_pR_fw_XbaI
2.5  µl T9002_lux_pR_rv_SpeI_BamHI_RBS
2.0  µl Template DNA (T9002 plasmid)
0.5  µl Phusion DNA Polymerase
31.5 µl H2O
-------
50.0 µl

program:

98 °C   30 sec
98 °C   10 sec
59 °C   30 sec
72 °C   45 sec
72 °C    5 min
 4 °C   constant

HisTag cloning of Colicins for purification

  • Amplification of Colicins 
10   µl 5x Phusion HF Buffer
1.0  µl 10mM dNTPs
2.5  µl colE1/E9_prot_fw_BamHI
2.5  µl colE1_prot_rv_HindIII/colE9_prot_rv_XmaI
2.0  µl Template DNA
0.5  µl Phusion DNA Polymerase
31.5 µl H2O
-------
50.0 µl

program:

98 °C   30 sec
98 °C   10 sec
59 °C   30 sec
72 °C   45 sec
72 °C    5 min
 4 °C   constant

[back]

Wednesday 09/10/2008

pSB1A3-Receiver-Colicin cloning

  • Gelextraction Receiver PCR: 0.7% Agarose, 100 V, 60 min
    080910 Rec ohne GFP small.jpg
  • Gelextraction Receiver PCR: QIAquick Gel Extraction (Qiagen), eluted in 30 µl H2O
  • Digestion of Receiver & pSB1A3 with SpeI and XbaI, 1 h 30 min 37 °C -> 10 min 65°C 
20 µl DNA
 6 µl H2O
 4 µl NEBuffer 2
 3 µl SpeI (NEB)
 3 µl XbaI (NEB)
 4 µl BSA 10x
-----
40 µl
  • Gelextraction of pSB1A3 digestion: 0.7 % Agarose, 50 min, 135 V
    080910 pSB1A3 gelex small.jpg
  • Gelextraction of pSB1A3 digestion: QIAquick Gel Extraction (Qiagen), eluted in 30 µl H2O, cutted pSB1A3 and T9002 -> not used
  • Ligation of pSB1A3 and Receiver: 1 h RT, 10 min 65 °C 
10 µl Receiver DNA
 2 µl Vector DNA pSB1A3 (sapped, from last week)
 2 µl Buffer T4 DNA Ligase (Fermentas)
 2 µl T4 DNA Ligase (Fermentas)
 4 µl H2O Nuclease Free (Fermentas)
-----
20 µl
  • Transformation of prior ligation: 5 µl per 50 µl E. coli MG1655 
- start thawing 50 µl competent E. coli MG1655 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 200 µl preheated LB media
- incubate at 37 °C for 1 h, 300 rpm
- Plate 200 µl on preheated LB-Amp plate
- incubate overnight
  • Controllgels of Colicin Amplification (Tuesday 080909): 1% Agarose, 37 min, 135V
    1. Colicin E1 & E1 for HisTag
      20080910 ColE1 His ColE1 His PCR080909 small.jpg
    2. Colicin E9 lys & E9 for HisTag
      20080910 ColE9 His ColE9 lys PCR080909 small.jpg
    3. Colicin E9 whole plasmid
      20080910 ColE9 PCR080909 small.jpg



HisTag cloning of Colicins for purification

  • Gelextraction of Colicin E1 and Colicin E9 PCR: 0,7%, 60 min, 100 V
    080910 colE1His colE9His gelex.jpg
  • Gelextraction of Colicin E1 and Colicin E9 PCR: QIAquick Gel Extraction (Qiagen), eluted in 30 µl H2O

Colicin activity test

  • inocculation of liquid cultures from glycerolstocks, incubation over night at 37 °C 
-10 ml M9 media + 10 µl Kanamycin + E. coli Top10 with I20260
-10 ml M9 media + 10 µl Ampicilin + E. coli MG1655 with pKC67 (Colicin E9 plasmid)
-10 ml M9 media + 10 µl Ampicilin + E. coli MG1655 with pKC67 (Colicin E9 plasmid)
-10 ml M9 media + 200 µl 10% Glucose + E. coli JC411 (Colicin E1 plasmid)
-10 ml M9 media + 200 µl 10% Glucose + E. coli JC411 (Colicin E1 plasmid)
-10 ml LB media + 200 µl 10% Glucose + E. coli JC411 (Colicin E1 plasmid)
-10 ml LB media + 200 µl 10% Glucose + E. coli JC411 (Colicin E1 plasmid)

[back]

Thursday 09/11/2008

pSB1A3-Receiver-Colicin cloning

  • Digestion of pSB1A3 with XbaI and SpeI from cloning cultures of last week. We used these cultures because the bande where easier to distinguish. !!!
  • 2008-09-18 UPDATE: the different bands were probably from the religated pSB1A3 plasmid, which cannot be cutted with XbaI and SpeI. -> No cutted pSB1A3 backbone
  • Gelextraction of pSB1A3: 0.7 % Agarose, 1 h, 135 V
    080911 colE9his vec and pSB1A3 digestion gelex small.jpg
  • Gelextraction of pSB1A3: QIAquick Gel Extraction (Qiagen), eluted in 30 µl H2O
  • Overnight ligation of pSB1A3(gelextraction, Thursday 2008-09-11) and T9002_without_GFP (gelextraction, Wednesday 2008-09-10) 
10 µl Receiver DNA
 2 µl Vector DNA pSB1A3
 2 µl Buffer T4 DNA Ligase (Fermentas)
 2 µl T4 DNA Ligase (Fermentas)
 4 µl H2O Nuclease Free (Fermentas)
-----
20 µl
  • Gelextraction colicin E1, colicin E9, colicin E9 lysis genes: 0.7 % Agarose, 1 h, 135 V
    20080911 PCR colicins gelex small.jpg
  • Gelextraction colicin E1, colicin E9, colicin E9 lysis genes: QIAquick Gel Extraction (Qiagen), eluted in 40 µl H2O -> stored ad -20 °C

HisTag cloning of Colicins for purification

  • Miniprep of pQE-30 plasmids from ON-culture(QIAprep Spin Miniprep Kit (250), Qiagen)
  • Digestion of pQE-30 (E1 -> HindIII, E9 -> XmaI) 
 20 µl DNA
 6 µl H2O
 4 µl NEBuffer 2/4
 3 µl BamHI (NEB)
 3 µl HindIII/XmaI (NEB)
 4 µl BSA 10x
-----
40 µl
  • Digestion of Colicin E1/E9 (E1 -> HindIII, E9 -> XmaI) 
 20 µl DNA
 6 µl H2O
 4 µl NEBuffer 2/4
 3 µl BamHI (NEB)
 3 µl HindIII/XmaI (NEB)
 4 µl BSA 10x
-----
40 µl
  • Gelextraction of pQE-30 and Colicin E1 digestion: 0.7 % Agarose, 1 h, 135 V
    080911 colE1His and vector digestion gelex small.jpg
  • Gelextraction of pQE-30 and Colicin E9 digestion: 0.7 % Agarose, 1 h, 135 V
    080911 colE9his vec and pSB1A3 digestion gelex small.jpg
  • Gelextraction of pQE-30 and Colicins digestion: QIAquick Gel Extraction (Qiagen), eluted in 30 µl H2O
  • Overnight ligation: 
10 µl Colicin DNA
 2 µl Vector DNA pQE-30
 2 µl Buffer T4 DNA Ligase (Fermentas)
 2 µl T4 DNA Ligase (Fermentas)
 4 µl H2O Nuclease Free (Fermentas)
-----
20 µl

Colicin activity test

  • 9 am: One E9 - M9 culture, one E1 - M9 culture and one E1 - LB culture were stressed with 10 µl of 0.1 mg/ml Mytomycin C
  • 4 pm: The colicin E1 LB cultures were thrown away, because the grew well in M9. The MG1655 and colicin cultures were sonicated in a ultrasonic bath three time for 15 seconds. After sonication cell extract was observed under microscope -> lot of dead cells -> succesfull lysis.
  • Overnight-Activity-Assay:
    • lysed cells were centrifuged for 15 min at 400 rpm. The supernatants were transferd to new falcons and were used for a colicin activity test.
    • well plate scheme:
      Platescheme small.jpg
3:1 -> 3 ml M9 media + 1 ml supernatant + 200 µl reference promotor cells (I20260)
1:1 -> 2 ml M9 media + 2 ml supernatant + 200 µl reference promotor cells (I20260)
1:3 -> 1 ml M9 media + 3 ml supernatant + 200 µl reference promotor cells (I20260)
    • ON-measurement for 14 h at 37 °C in Tecan well plate reader

[back]

Friday 09/12/2008

pSB1A3-Receiver-Colicin cloning

  • New ligation because controll gel didn't show any bands for pSB1A3 -> probably a too low concentration: : 1 h 40 min RT, 15 min 65 °C 
10 µl Receiver DNA
 6 µl Vector DNA pSB1A3 (sapped, from last week)
 2 µl Buffer T4 DNA Ligase (Fermentas)
 2 µl T4 DNA Ligase (Fermentas)
-----
20 µl
  • Transformation of ligation (Tuesday 2008-09-11) into E. coli TOP 10 cells: 5 µl per 50 µl E. coli BL21 cells 
- start thawing 50 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 200 µl preheated LB media
- incubate at 37 °C for 1 h, 300 rpm
- Plate 200 µl on preheated LB-Amp plate
- incubate overnight at 37 °C
  • Controllgel of PCR: 1% Agarose, 30 min, 135 V
    080912 controllgel PCR small.jpg

HisTag cloning of Colicins for purification

  • Transformation of ColE1 and ColE9 into BL21 cells: 5 µl per 50 µl E. coli BL21 cells 
- start thawing 50 µl competent E. coli BL21 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 200 µl preheated LB media
- incubate at 37 °C for 1 h, 300 rpm
- Plate 200 µl on preheated LB-Amp plate
- incubate overnight at 37 °C

Colicin activity test

  • Colicin E1 and E9 kills other E. coli cells efficient
11092008-igem colicin activity sonicbath on OD 3-1 beschriftet.jpg
11092008-igem colicin activity sonicbath on GFP 3-1 beschriftet.jpg
11092008-igem colicin activity sonicbath on OD 1-1 beschriftet.jpg
11092008-igem colicin activity sonicbath on GFP 1-1 beschriftet.jpg
11092008-igem colicin activity sonicbath on OD 1-3 beschriftet.jpg
11092008-igem colicin activity sonicbath on GFP 1-3 beschriftet.jpg

Other

  • Maxiprep of I15030 and T9002 for plasmid backbone. (Compact Prep Plasmid Maxi Kit (25), Qiagen)

[back]

Saturday 09/13/2008

pSB1A3-Receiver-Colicin cloning

  • Transformation results: we picked and plated 8 colonies of each transformation on LB-Ampicilin plates and in addition we inocculated liquid cultures.

HisTag cloning of Colicins for purification

  • Transformation results: We plated our transformation on LB-Kanamycin because BL21 cells contain a helper plasmid which has an Kanamycin resistance. But pQE-30 plasmids have an Ampicilin resistance. Because of that we transderd colonies from an bacteria lawn to Ampicilin/Kanamycin plates.



Sunday 09/14/2008

pSB1A3-Receiver-Colicin cloning

  • Miniprep of pSB1A3-Receiver-Cloning from ON-cultures(QIAprep Spin Miniprep Kit (250), Qiagen)
  • Digestion of pSB1A3-Rec XbaI and SpeI, 1h 30 min -> 37 °C, 15 min -> 65 °C 
10  µl DNA
3   µl H2O
2   µl NEBuffer 2
1.5 µl XbaI (NEB)
1.5 µl SpeI (NEB)
2   µl BSA 10x
------
20  µl
  • Gel for selection of positive clones: Estimated fragments ~2150 bp pSB1A3 backbone and ~1088 bp T9002 without GFP. No bands at 1088 bps. Colony-PCR-Screen on Mondayday.
  • Since selection of positive clones failed, we started a new ligation
  • Sapping: 30 min 37°C, 10 min 65°C 
34 µl pSB1A3 (digested)
 4 µl SAP Buffer
 1 µl SAP Enzyme
-----
39 µl
  • Ligation of pSB1A3 and T9002 without GFP: 18 h 16 °C, 20 min 65 °C, 4 °C const 
 6 µl Receiver DNA
 4 µl Vector DNA pSB1A3
 2 µl Buffer T4 DNA Ligase (NEB)
 2 µl T4 DNA Ligase (NEB)
 6 µl H2O
-----
20 µl

HisTag cloning of Colicins for purification

  • Plated colonies from Saturdayurday (2008-09-13) were not grown.
  • Retransformation of ligation:
    • ColE1His: 2.5 µl ligation on 50 µl BL-21 cells, 10 µl ligation on 100 µl TOP 10 cells
    • ColE9His: 2.5 µl ligation on 50 µl BL-21 cells, 10 µl ligation on 100 µl TOP 10 cells
  • New Ligation with other insert/plasmid ration
  • Ligation of pQE-30 and Colicins: 18 h 16 °C, 20 min 65 °C, 4 °C const 
 9 µl ColE1/E9 His
 3 µl Vector DNA pQE-30
 2 µl Buffer T4 DNA Ligase (NEB)
 2 µl T4 DNA Ligase (NEB)
 4 µl H2O
-----
20 µl

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