Team:Heidelberg/Notebook/Killing II/11thweek

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Latest revision as of 21:50, 28 October 2008

11^th week

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Monday 10/13/2008

Construction of reporterplasmid with kanamycin resistance

mCherry

• Transformationresults: Cultures were grown, also on plates with wrong plasmid - insert ratio
• Inoculation of liquid cultures from transformation with wrong ratio in the morning.
• Miniprep of liquid cultures in the evening: eluted in 35 µl H₂O, (Qiagen Miniprepkit)
• Inoculation of liquid cultures from transformation with right ratio.

pSB1A2-Receiver-Colicin cloning

Mutagenesis of pSB1A2-ColicinE1-Receiver

• MiniPreps of colE1 after the 2nd mutagenesis (colonies 1.14, 1.16, 5.1, 5.7) and of colE9 (colonies 2 and 4, still with the pre-/suffix) each on pSB1A2 after receiver --> sent for sequencing
• Colonies picked from the transformations of the mutated colE9 parts (with the correct pre-/suffix) --> inocculated and MiniPreped (for digestion and sequencing)

Characterization: ColicinE1-Receiver Activitytest

• 4 pm: Inoculation of:
  • 30 ml TB-Kana with BBa_I20260
  • 30 ml TB-Amp with T9002-Receiver without GFP
  • 30 ml TB-Amp with pSB1A2-ColicinE1-Receiver
• 8.30 pm: Preparing mixtures for the plate:
  • plate scheme:
    
  • Killer:Prey 1:0.25 -> 200 µl Referencepromotercells + 800 µl Receivercells
  • Killer:Prey 1:1 -> 200 µl Referencepromotercells + 200 µl Receivercells + 600 µl TB media
  • Killer:Prey 1:5 -> 200 µl Referencepromotercells + 40 µl Receivercells + 760 µl TB media
  • Killer:Prey 1:10 -> 200 µl Referencepromotercells + 20 µl Receivercells + 780 µl TB media
• 9.30 pm: Starting measurement

Sender cloning: constitutive promotor-sender

Characterization: Sender Activity Test

• Characterization: Sender-Test measured every hour over the day. See Saturdayurday for more details.
• Layout of the 96 well plate:
  
  
• Results:
  • AI-1 GFP/OD:
    
    
  • AI-1 OD:
    
    
  • Sender GFP/OD:
    
    
  • Sender OD:
    
    
  • Amplifier GFP/OD:
    
    
  • Amplifier OD:
    
    
  • In most cases the later supernatants have a higher GFP/OD rate: But there in some cases earlier concentrations are much higher. A problem was that the plate stand at 4 °C for 2 days so this could cause these outliers. In the next days we repeat this experiment to characterize these parts properly.

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Tuesday 10/14/2008

Construction of reporterplasmid with kanamycin resistance

mCherry

• Controldigestion of Cloning: 2 h 20 min -> 37 °C

10.0 µl pBAD-mCherry DNA (3rd mutation)
 4.0 µl BSA 10x (NEB)
 4.0 µl NEBuffer 4 (NEB)
 0.5 µl SacI (NEB)
 0.7 µl XbaI (NEB)
20.8 µl H2O
-------
40.0 µl

• Gel of digestion: 1% Agarose, 135 V, 30 min
  
• Results of Digestion:
  • Expected fragments for correct mutation: ~1100 bp & ~4600 bp
  • Coolonies 1, 2, 4 & 5 shows the right bands. -> Sucessful cloning.

pSB1A2-Receiver-Colicin cloning

Mutagenesis of pSB1A2-Receiver-ColicinE1

• Miniprep of ON liquid cultures 3rd colicin E1 mutagenesis: eluted in 35 µl H₂O, Qiagen Miniprepkit
• Controldigestion of 3rd Mutagenesis: 2 h 20 min -> 37 °C

10.0 µl ColE1-Receiver DNA (3rd mutation)
 4.0 µl BSA 10x (NEB)
 4.0 µl NEBuffer 3 (NEB)
 0.5 µl PstI (NEB)
21.5 µl H2O
-------
40.0 µl

• Gel of digestion: 1% Agarose, 135 V, 30 min
  
• Results of Digestion:
  • Expected fragments for correct mutation: ~695 bp & ~4600 bp
  • The gel shows the right bands. This indicates that our three mutations were succesful.
• Mutagenesis PCR of Miniprep: Mutagenesis of 3rd PstI site

 5.0  µl pfu buffer
 1.0  µl forward primer (10 µM Col E1_mut_Pst_2_fw)
 1.0  µl reverse primer (10 µM Col E1_mut_Pst_2_rv)
 1.0  µl dNTPs (12.5 mM, 50x)
 0.5  µl template DNA - 5 to 50 ng plasmid DNA
40.5 µl milliQ water
 1.0  µl turbo pfu polymerase (Stratagene)
 ----
 50.0 µl

95 °C   30 sec
95 °C   30 sec    |
55 °C   60 sec    | 16 cycles
68 °C   11 min    |
 4 °C   constant

• Digestion with DpnI (NEB) of the third mutagenesis to cut parental plasmid: Added 1 µl of DpnI to the finished Mutagenesis PCR. DpnI cuts at methylated GATC sites, so that only parental plasmids without the mutation are cutted. 2 h 35 min -> 37 °C
• Controlgel of third Mutagenesis (PstI_3): 1 % Agarose, 135 V, 30 min; DNA only visible in products from the colonie 1 (1.14 and 1.16) -> probably the mutagenesis worked on this plasmid.
  
• PCR Purification Kit (Qiagen) to purify the plasmids. Eluted in 35 µl H₂O
• Transformation of prior mutagenesis: 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 500 µl on preheated LB-Amp plate
- incubate overnight

Characterization: ColicinE1-Receiver Activitytest

• Results:
  • Killer:Prey ratio 1:0.25 GFP:
    
    
  • Killer:Prey ratio 1:0.25 OD:
    
    
  • Killer:Prey ratio 1:1 GFP:
    
    
  • Killer:Prey ratio 1:1 OD:
    
    
  • Killer:Prey ratio 1:5 GFP:
    
    
  • Killer:Prey ratio 1:5 OD:
    
    
  • Killer:Prey ratio 1:10 GFP:
    
    
  • Killer:Prey ratio 1:10 OD:
    
    

Sender cloning: constitutive promotor-sender

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Wednesday 10/15/2008

Construction of reporterplasmid with kanamycin resistance

mCherry

• Sequencing results: Colonies 1, 2, 4 & 5 are positive clones. -> double transformation

pSB1A3-Receiver-Colicin cloning

Standardization

• Inoculation of 4th mutagenesis transformation cultures
• Miniprep of ColE9-Receiver BioBrickstandard: 5 colonies of ColE9-Receiver(2) and 5 colonies of ColE9-Receiver(4) (Qiagen, Miniprepkit) --> all 10 sent for sequencing

Activity Test

• Results

Sender

• Sendertest

Visualization

• Doubletransformation of:

I20260 + constitutive sender in TOP 10
I20260 in TOP 10
I20260 + ColicinE1-Receiver in TOP 10
I20260 + T9002_without_GFP-Receiver in TOP 10
pBAD-mCherry + ColicinE1-Receiver in TOP 10
pBAD-mCherry + T9002_without_GFP-Receiver in TOP 10
pBAD-mCherry + ColicinE1-Receiver in HCB33
pBAD-mCherry + T9002_without_GFP-Receiver in HCB33
pBAD-mCherry + ColicinE1-Receiver in MG1655
pBAD-mCherry + T9002_without_GFP-Receiver in MG1655

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Thursday 10/16/2008

pSB1A2-Receiver-Colicin cloning

Sequencing results of ColE9-Receiver cloning

• Miniprep of ONC

• Controldigestion of Minipreps:
• Gel of Digestion:
• PCR of Minipreps:

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

98 °C    2 min
98 °C   10 sec   |
53 °C   30 sec   | 25 cycles
72 °C   90 sec   |
72 °C    5 min
 4 °C   constant

• Gel of PCR
• Gelextraktion
• Ligation ColicinE1-Receiver (BioBrick Standard) with pSB1A2 (3:1): 1 h -> 22 °C

 2.0 µl T4 DNA Ligase (Fermentas)
 2.0 µl T4 DNA Ligase Buffer (Fermentas)
 2.6 µl pSB1A2 (6,5 ng/µl)
13.4 µl ColE1Receiver PCR Product (5,6 ng/µl)

• Transformation of prior ligation: 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 500 µl on preheated LB-Amp plate
- incubate overnight

Colicin activity test

After adjustment of the OD of the different cultures (killer, reference killer, prey), mixtures of different rations were done and transfered to a 96 well plate for ON measurement after following schemes

Sender

• Results of sender test

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Friday 10/17/2008

pSB1A3-Receiver-Colicin cloning

HisTag cloning of Colicins for purification

Sender Cloning: pBAD - sender

Sender Cloning: constitutive promotor - sender

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Saturday 10/18/2008

pSB1A3-Receiver-Colicin cloning

ColE1 Cloning for standardization

• PCR-Amplification of Receiver-Col E1-Insert with the right Prefix and Suffix

25.0 µl Taq Master Mix (Fermentas)
 2.5 µl T9002_LuxpR_Not_Eco_Xba_G_fw (Tm=80,96°C)
 2.5 µl ColE1_kil_prot_rv_A_SpeI (Tm=66,94°C)
20.0 µl H2O
   1    colony
-------
50.0 µl

program 1:

98 °C    10 sec
98 °C    30 sec         |
57 °C    30 sec         | 25 cycles
72 °C    1 min 45 sec   |
72 °C    10 min
 4 °C   constant

program 2:

98 °C    10 sec
98 °C    30 sec         |
67 °C    30 sec         | 25 cycles
72 °C    1 min 45 sec   |
72 °C    10 min
 4 °C   constant

• Gel of Receiver-colE1 PCR-amplification: 0,7% Agarose, 135 V, 30 min
• Gel-Results of colE1 PCR-amplification: Bands of PCR-A
• Gelextraction with Qiagen Kit: eluted in 40 µl H2O

• Digestion of receiver-colicinE1-insert for 2 h at 37°C

38.5  µl DNA (of Gelextraction)
 0.5 µl EcoRI (20 000 U/ml, NEB)
 1.0 µl SpeI (10 000 U/ml, NEB)
 5.0 µl BSA 10x
 5.0 µl EcoRI Buffer(NEB)
-------
50.0 µl

• PCR-Purification
• Ligation of receiver-colE1-insert into pSB1A2 (1):

 1.Ligation attempt (+):
 2.0 µl T4 DNA Ligase Buffer (Fermentas)
 2.0 µl T4 DNA Ligase (Fermentas)
 1.8 µl pSB1A2
14.2 µl receiver-colE1-insert (From PCR-Purification)
-------
20.0 µl


2.Ligation attempt (-):
 2.0 µl T4 DNA Ligase Buffer (Fermentas)
 2.0 µl T4 DNA Ligase (Fermentas)
 1.1 µl pSB1A2
14.9 µl receiver-colE1-insert (From PCR-Purification)
-------
20.0 µl

• Transformation of receiver-colE1-pSB1A2 BioBrick: 5 µl DNA (Ligation) per 50 µl E. coli TOP 10 competent 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 250 µl preheated LB media
- incubate at 37 °C for 20 min, 500 rpm
- Plate 300 µl on preheated LB-Amp plate
- incubate overnight at 37°C

Sender Cloning: constitutive promotor - sender

Sender and Amplifier Activity Test - 24h

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Sunday 10/19/2008

pSB1A3-Receiver-Colicin cloning

ColE1 Cloning for standardization

• Inoculation of colonies from transformation:
  • 5 colonies from each plate in 10 ml LB-amp medium respectively:
  • 57°C(+)-1
  • 57°C(+)-2
  • 57°C(-)-1
  • 57°C(-)-2
  • 67°C(+)-1
  • 67°C(+)-3
  • 67°C(-)-1
  • 67°C(-)-3
• Minipreps:
  • 57°C(+)-1
  • 57°C(+)-2
  • 57°C(-)-1
  • 57°C(-)-2
  • 67°C(+)-1
  • 67°C(+)-3
  • 67°C(-)-1
  • 67°C(-)-3
• Controldigestion with XbaI, SpeI and XbaI+SpeI of BioBrick Cloning: Receiver-ColicinE1-pSB1A3

XbaI digestion: 
10.0 µl DNA
 2.0 µl BSA 10x (NEB)
 2.0 µl NEBuffer 2
 0.5 µl XbaI (NEB) 
 5.5 µl H2O
-------
20.0 µl

SpeI digestion:
10.0 µl DNA
 2.0 µl BSA 10x (NEB)
 2.0 µl NEBuffer 2
 1.0 µl SpeI (NEB) 
 5.0 µl H2O
-------
20.0 µl

SpeI digestion:
10.0 µl DNA
 2.0 µl BSA 10x (NEB)
 2.0 µl NEBuffer 2
 0,5 µl XbaI
 1.0 µl SpeI (NEB) 
 4.5 µl H2O
-------
20.0 µl

• Controlgel: 0,7% Agarose, 135 V, 30 min
• Gelresults:
  • Expected Fragments:
    • XbaI digestion (X): ~5200 bp (linear plasmid)
    • SpeI digestion (S): ~5200 bp (linear plasmid)
    • XbaI+SprI digestion (X+S): ~3200 bp (Insert) + ~2000 bp (Vector)
    • Undigested Plasmid (P): ~3200 bp

• • The following clones are positive ColE1 BioBricks:
    • 57°C(+)-1
    • 57°C(+)-2
    • 57°C(-)-1
    • 57°C(-)-2
    • 67°C(+)-1
    • 67°C(-)-1

Colicin Activity Test

Sender Cloning: constitutive promotor - sender

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