Team:Heidelberg/Notebook/Killing II/4thweek

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Latest revision as of 21:25, 28 October 2008

4^th week

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Monday 08/25/2008

Colicin Receiver

pSB1A3-Receiver-Colicin-Cloning

PCR of colicin E1 and E9 operon with specific primers:

25.0 µl Phusion MasterMix
 2.5 µl Primer fw(ColE1_prot_fw_BamHI/ColE9_plasmid_rv_SpeI)
 2.5 µl Primer rv(ColE1_kil_prot_rv_SpeI/ColE9_plasmid_rv_SpeI)
17.0 µl H₂O
 3.0 µl pColE1/pColE9-J
-------
50.0 µl

program:
98 °C   30 sec
98 °C   10 sec   |
57 °C   20 sec   | 25 cycles 
72 °C   45 sec   |
72 °C    8 min
 4 °C   constant

Activity Test

Inoculation of 5 ml liquid ONC with...
- pColE1 in JC411 -> 0.1% Glucose Medium
- pColE9 in MG1655
- TOP 10
- MG 1655

Sender part

pBAD-Sender Cloning

Controldigestion of BBa_F1610 with DraI: 1.5 - 2 h -> 37 °C

10 µl DNA (320-330 ng/µl)
 3 µl Tango Buffer 10x (Fermentas)
 2 µl DraI (Fermentas)
15 µl H₂O
-----
30 µl

Gel of digestion: Results are not perfect but expected bands can be estimated.

Activity Test

Inoculation of pBAD-BBa_F1610 in 8 ml M9 Kana + 0.1% Arabinose. (8 colonies)

General

Inoculation of liquid ONC of BBa_J23107, BBa_J23102, BBa_R0011, BBa_R0040 in 5 ml LB Amp. (For Minipreps and Glycerolstocks)

Tuesday 08/26/2008

Colicin Receiver

pSB1A3-Receiver-Colicin-Cloning

PCR Purification of T9002 without GFP (08/22/08) and colicin E1/E9 operon: Qiagen PCR Purification Kit.
Digestion of purified products with BamHI: 2h -> 37 °C

20.0 µl DNA (PCR product, purified)
13.0 µl H₂O
 4.0 µl NEBuffer 3 (NEB)
 3.0 µl BamHI (NEB)
 4.0 µl BSA 10x (NEB)
-------
44.0 µl

Gelextraction of digestion: Cutted the bands and freezed for purification tomorrow.

Activity Test

2nd step of colicin test based on ONC from Tuesdaysday:
- pColE1 (JC411):
  - inoculation of 12 ml LB ONC + 0.1% Glucose -> unstressed
  - inoculation of 12 ml LB ONC + 0.1% Glucose + 1µg/ml Mytomycin C
  - EDIT 09/02/08: Too high Mytomycin C concentrations
  - 100 µl ONC + 300 µl LB + 200 µl 10% glucose plated on LB-Agar
- pColE9 (MG1655):
  - inoculation of 12 ml LB ONC-> unstressed
  - inoculation of 12 ml LB ONC + 1µg/ml Mytomycin C
  - EDIT 09/02/08: Too high Mytomycin C concentrations
  - 100 µl ONC + 500 µl LB plated on LB-Agar
- MG1655 + TOP10:
  - inoculation of 12 ml LB ONC-> unstressed
  - 100 µl ONC + 500 µl LB plated on LB-Agar

Sender part

pBAD-Sender Cloning

Control of Cloning -> see activity test

Activity Test

To check if we have one positive clone we perform the sender activity test again (see Fridayday 08/08/22 for plate scheme and details). After the first measurements an increase in GFP expression by BBa_T9002 was observed for colonies 9, 10, 11. Because of that we inoculated liquid ONC with LB-Kana for miniprepes and glycerolstocks

Wednesday 08/27/2008

Colicin Receiver

pSB1A3-Receiver-Colicin-Cloning

Gelextraction of BamHI digestion: Qiagen Gelextraction kit
Ligation of receiver with colicins ratio 1:1: 1h -> RT, 10 min -> 65 °C

10 µl DNA I
10 µl DNA II
 4 µl 10x T4 DNA Ligase Buffer
 2 µl T4 Ligase
14 µl H₂O
-----
40 µl

Gelextaction of Ligation: ligated construct (~3000 bp) was extracted and purified. Qiagen Gel Extraction Kit
Digestion of ligated product with XbaI and SpeI: 1.5 h -> 37 °C and 65 °C -> 20 min

20 µl DNA
 6 µl H₂O
 4 µl NEBuffer 2
 3 µl SpeI
 3 µl XbaI
 4 µl BSA 10x
-----
40 µl

Gel of E1 and E9 digestion: no fragments were visible
Gel of BBa_T9002 digestion: Expected bands cannot be differentiated because fragment sizes are too similar ~1950 bp and ~2150 bp.
Send probes of pColE9 (colE9_prot_fw_XbaI), pColE1(colE1_prot_fw_XbaI/colE1_prot_rv_SpeI/colE1_imm_fw_SpeI) and BBa_T9002 (pSB_ins_fw/pSB_ins_rv) to GATC for sequencing.

Activity Test

Creation of Supernatant of stressed and unstressed colicin cultures.
Plate the 4 different supernatants and the 4 different cultures on the prepared ColE1, ColE9, MG1655 and TOP10 plates.

Sender part

pBAD-Sender Cloning

Miniprep and Glycerolstocks of pBAD18-BBa_F1610 colony 9, 10, 11
Send miniprep of pBAD18-BBa_F161 cloning colony 9 (VIC121/VIC122 align on pBAD18)to GATC for sequencing.

Activity Test

24h measurement of Colicin test

Thursday 08/28/2008

Colicin Receiver

pSB1A3-Receiver-Colicin-Cloning

Sequencing results: ColE1_prot correct. Others were not sequenced.
New attempt of second digestion: no bands are visible.
New cloning strategy:
1. Cloning of AHL-receiver without GFP into pSB1A3 (Amplification from BBa_T9002 with T9002_XbaI_fw and T9002_SpeI_BamHI_RBS_rv primers)
2. Cloning of colicin operon behind LuxPr promoter.
- In addtion:
PCR of BBa_T9002 with new primers standardprotocol Phusion MasterMix (Finnzymes, NEB)

25.0 µl Phusion MasterMix
 2.5 µl Primer fw
 2.5 µl Primer rv
 2.0 µl DNA Template
18.0 µl H₂O
-------
50.0 µl

PCR Purification and Analytical gel -> right fragment size
Digestion of PCR product and vector with XbaI and SpeI
Gelextraction: expected bands (~1061 bp LuxpR-receiver and ~2157 bp pSB1A3-backbone) could be separated and were eluted in 34 µl H₂O.
Sapping of vector to avoid self-ligation: 30 min 37 °C -> 15 min 65 °C

34.0 µl pSB1A3 DNA (XbaI/SpeI cutted)
 4.0 µl SAP Buffer (Fermentas)
 1.0 µl SAP enzymes (Fermentas)

Ligation: 16 °C -> Overnight

 6.0 µl Receiver DNA
 2.0 µl Vector DNA
 2.0 µl Buffer T4 DNA Ligase
 8.0 µl H₂O
-------
20.0 µl

Activity Test

Results: No effects were obtained
Preparation of probes for inocculation over night to measure colicin E1 activity:
- 1 x TOP 10
- 1 x TOP 10 + 2 ml ColE1 supernatant unstressed
- 1 x TOP 10 + 2 ml ColE1 supernatant stressed
- 1 x TOP 10 + 2 ml ColE9 supernatant unstressed
- 1 x TOP 10 + 2 ml ColE9 supernatant stressed
- 1 x TOP 10 + 2 ml MG1655

Sender part

pBAD-Sender Cloning

Sequencing results: pBAD18-BBa_F1610 is the right construct.

Friday 08/29/2008

Colicin Receiver

pSB1A3-Receiver-Colicin-Cloning

Transformation of ON-Ligation

Activity Test

Results:

OD Measurement of colicin treated ONCs. An effect in cultures of Mytomycin C stressed cells were observed. But this could also be the toxic effect of Mytomycin C. In addition we obtained that we used a too high concentration of Mytomycin C (1 µg/ml instead of 0.1 µg/ml).
Measured cell growth curve over night: Logistic growth could be observed but in general growth curve does not look that good.

Sender part

General

Seminar on Synthetic Biology: Chris
Breakfast
Project Presentation for Frankfurter Allgemeine Zeitung
Campus TV
Team Meeting

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