Team:Heidelberg/Notebook/Killing II/6thweek

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Revision as of 15:46, 27 October 2008

6^th week

go back to 5^th week

go back to the overview

Monday 09/08/2008

pSB1A3-Receiver-Colicin cloning

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

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

Wednesday 09/10/2008

pSB1A3-Receiver-Colicin cloning

Gelextraction Receiver PCR: 0.7% Agarose, 100 V, 60 min
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
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
2. Colicin E9 lys & E9 for HisTag
3. Colicin E9 whole plasmid

HisTag cloning of Colicins for purification

Gelextraction of Colicin E1 and Colicin E9 PCR: 0,7%, 60 min, 100 V
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)

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
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
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
Gelextraction of pQE-30 and Colicin E9 digestion: 0.7 % Agarose, 1 h, 135 V
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:

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

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

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)

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

go to 7^th week

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+                            Keep it sync with the drop-down menu width. */
+}
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1,
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM1 li a,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM1 li a {
+  width: 10em; /* Side menu width. */
+}
+/*...............................................................................................*/
+/*
+Menu Bar 1
+Drop-Down Menu #2
+Side Menu #2
+*/
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2 {
+  left: 15.5em  !important; /* Places the side menu to the right of the drop-down menu.
+                            Keep it sync with the drop-down menu width. */
+}
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2,
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM2 li a,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM2 li a {
+  width: 10em; /* Side menu width. */
+}
+/*...............................................................................................*/
+/*
+Menu Bar 1
+Drop-Down Menu #2
+Side Menu #3
+*/
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3 {
+  left: 15.5em  !important; /* Places the side menu to the right of the drop-down menu.
+                            Keep it sync with the drop-down menu width. */
+}
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3,
+div.MenuBar#navi ul li:hover ul.DropDownMenu li:hover ul.SideMenu#MB1-DDM2-SM3 li a,
+div.MenuBar#navi ul li a:hover ul.DropDownMenu li a:hover ul.SideMenu#MB1-DDM2-SM3 li a {
+  width: 10em; /* Side menu width. */
+}
+/*...............................................................................................*/
+</style>
 <body>

Team:Heidelberg/Notebook/Killing II/6thweek

From 2008.igem.org

Revision as of 15:46, 27 October 2008

Contents

Monday 09/08/2008

pSB1A3-Receiver-Colicin cloning

Tuesday 09/09/2008

pSB1A3-Receiver-Colicin cloning

HisTag cloning of Colicins for purification

Wednesday 09/10/2008

pSB1A3-Receiver-Colicin cloning

HisTag cloning of Colicins for purification

Colicin activity test

Thursday 09/11/2008

pSB1A3-Receiver-Colicin cloning

HisTag cloning of Colicins for purification

Colicin activity test

Friday 09/12/2008

pSB1A3-Receiver-Colicin cloning

HisTag cloning of Colicins for purification

Colicin activity test

Other

Saturday 09/13/2008

pSB1A3-Receiver-Colicin cloning

HisTag cloning of Colicins for purification

Sunday 09/14/2008

pSB1A3-Receiver-Colicin cloning

HisTag cloning of Colicins for purification