Team:Braunschweig/Protocols

From 2013.igem.org

(Difference between revisions)
Line 87: Line 87:
10 g Bacto yeast extract<br>
10 g Bacto yeast extract<br>
5 g NaCl<br>
5 g NaCl<br>
-
were dissolved in 1 L DI water, the pH was adjusted to 7.0.<br>
+
were dissolved in 1 L dH<sub>2</sub>O, the pH was adjusted to 7.0.<br>
For solid medium 12 g of Bacto agar (per Liter) was added.<br><br>
For solid medium 12 g of Bacto agar (per Liter) was added.<br><br>
Line 94: Line 94:
51,1 mL glacial acetic acid (17,4M)<br>
51,1 mL glacial acetic acid (17,4M)<br>
200 mL 100mM EDTA (pH 8)<br>
200 mL 100mM EDTA (pH 8)<br>
-
were dissolved in 1L DI water.<br><br>
+
were dissolved in 1L dH<sub>2</sub>O.<br><br>
<b>Loading buffer for gel electrophoresis</b><br>
<b>Loading buffer for gel electrophoresis</b><br>
Line 101: Line 101:
<b>Ampicillin stock solution</b><br>
<b>Ampicillin stock solution</b><br>
-
10 g Ampicillin sodium salt was dissolved in 100 mL DI water, sterile filtered, aliquoted and stored at -20 °C.<br><br>
+
10 g ampicillin sodium salt was dissolved in 100 mL dH<sub>2</sub>O, sterile filtered, aliquoted and stored at -20 °C.<br><br>
<b>Chloramphenicol stock solution</b><br>
<b>Chloramphenicol stock solution</b><br>
-
3.4 g Chloramphenicol was dissolved in 100 mL Ethanol, sterile filtered, aliquoted and stored at -20 °C.<br><br>
+
3.4 g chloramphenicol was dissolved in 100 mL Ethanol, sterile filtered, aliquoted and stored at -20 °C.<br><br>
<b>Clavulanic acid</b><br>
<b>Clavulanic acid</b><br>
-
1 g Clavulanic acid was dissolved in 100 mL DI water, sterile filtered aliqoted and stored at -20 °C.<br><br>
+
1 g clavulanic acid was dissolved in 100 mL dH<sub>2</sub>O, sterile filtered aliqoted and stored at -20 °C.<br><br>
<b>Tetracyclin stock solution</b><br>
<b>Tetracyclin stock solution</b><br>
Line 113: Line 113:
<b>TFB 1, pH 5.7 for preparation of competent cells</b><br>
<b>TFB 1, pH 5.7 for preparation of competent cells</b><br>
-
0.3 g Calciumchloride, 0.6 g Potassiumacetate, 1.2 g Rubidiumchloride, 1 g Manganesechloride x 4 H<sub>2</sub>O and 15 mL Glycerin were dissolved in DI water and diluted to a final volume of 100 mL. The pH was adjusted and the solution sterile filtered.<br><br>
+
0.3 g calciumchloride, 0.6 g potassiumacetate, 1.2 g rubidiumchloride, 1 g manganesechloride x 4 H<sub>2</sub>O and 15 mL glycerin were dissolved in dH<sub>2</sub>O and diluted to a final volume of 100 mL. The pH was adjusted and the solution sterile filtered.<br><br>
<b>TFB 2, pH 8.0 for preparation of competent cells</b><br>
<b>TFB 2, pH 8.0 for preparation of competent cells</b><br>
-
2.2 g Calciumchloride, 0.12 g Rubidiumchloride, 0.21 g Morphoslinopropanesulfuricacid and 15 mL Glycerin were dissolved in DI water and diluted to a final volume of 100 mL. The pH was adjusted and the solution sterile filtered.<br><br>
+
2.2 g calciumchloride, 0.12 g rubidiumchloride, 0.21 g morphoslinopropanesulfuricacid and 15 mL glycerin were dissolved in dH<sub>2</sub>O and diluted to a final volume of 100 mL. The pH was adjusted and the solution sterile filtered.<br><br>
<b>N-(butyryl)-homoserine lactone stock solution</b><br>
<b>N-(butyryl)-homoserine lactone stock solution</b><br>
-
1.7 mg N-butyryl-homoserine lactone was dissolved in 1 mL Dimethyl sulfoxide.The solution was sterile filtered.<br><br>
+
1.7 mg N-butyryl-homoserine lactone was dissolved in 1 mL dimethyl sulfoxide. The solution was sterile filtered.<br><br>
<b>N-(3-oxododecanoyl)-homoserine lactone</b><br>
<b>N-(3-oxododecanoyl)-homoserine lactone</b><br>
-
2.8 mg N-3-oxododecanoyl-homoserine lactonce was dissolved in 933 µL Dimethyl sulfoxide. The solution was sterile filtered.
+
2.8 mg N-3-oxododecanoyl-homoserine lactonce was dissolved in 933 µL dimethyl sulfoxide. The solution was filter sterilized.</p>
-
</p>
+
    
    
  </div>
  </div>
Line 143: Line 142:
<div id="Cultivation" class="menuSection">
<div id="Cultivation" class="menuSection">
-
     <h2><a href="#Cultivation">E. Coli Culture Conditions</a></h2>
+
     <h2><a href="#Cultivation"><i>E. Coli</i> Culture Conditions</a></h2>
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:0px;">For the cultivation of <i>E.coli</i> 2xYT medium was used. If necessary 1µL/mL of an antibiotic stock solution was added. The cultures were incubated at 37 °C and in case of liquid cultures shaken at 350 rpm.</p>
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:0px;">For the cultivation of <i>E.coli</i> 2xYT medium was used. If necessary 1µL/mL of an antibiotic stock solution was added. The cultures were incubated at 37 °C and in case of liquid cultures shaken at 350 rpm.</p>
    
    
Line 149: Line 148:
<div id="Conti" class="menuSection">
<div id="Conti" class="menuSection">
-
     <h2><a href="#Conti">E. Coli Continuous Cultivation</a></h2>
+
     <h2><a href="#Conti"><i>E. Coli</i> Continuous Cultivation</a></h2>
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">Continuous cultivations were conducted in a 2 L stirred tank bioreactor system (Applikon) with one six-bladed disc turbine impeller. Bioreactors were inoculated with mixed cultures which were derived from mixing different ratios of overnight monocultures to an OD<sub>520</sub> of 0.5. Growth temperature (37±0.1°C), aeration rate (2.5 L min<sup>−1</sup>), agitation speed (350 min<sup>-1</sup>) and pH value (pH 7.0) were automatically kept constant. The working volume was 1 L and the dilution rate was adjusted to  0.5 h<sup>-1</sup>.<br>
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">Continuous cultivations were conducted in a 2 L stirred tank bioreactor system (Applikon) with one six-bladed disc turbine impeller. Bioreactors were inoculated with mixed cultures which were derived from mixing different ratios of overnight monocultures to an OD<sub>520</sub> of 0.5. Growth temperature (37±0.1°C), aeration rate (2.5 L min<sup>−1</sup>), agitation speed (350 min<sup>-1</sup>) and pH value (pH 7.0) were automatically kept constant. The working volume was 1 L and the dilution rate was adjusted to  0.5 h<sup>-1</sup>.<br>
-
Depending on the mode of growth (regulated or unregulated) the medium was supplemented with Ampicillin or Chloramphenicol, respectively. Samples were taken every hour to monitor the cell density. To determine the ratios of the two strains in the culture, samples were spread out on agar plates, incubated at 37 °C and obtained colored colonies were counted.
+
Depending on the mode of growth (regulated or unregulated) the medium was supplemented with ampicillin or chloramphenicol, respectively. Samples were taken every hour to monitor the cell density. To determine the ratios of the two strains in the culture, samples were spread out on agar plates, incubated at 37 °C and obtained colored colonies were counted.
</p>
</p>
    
    
Line 160: Line 159:
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">Overnight cultures in 20 mL 2xYT medium containing chloramphenicol were inoculated directly from -80°C glycerol cell stock. Cells were grown in non-baffled-flasks at 37°C and 250 rpm.<br>  
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">Overnight cultures in 20 mL 2xYT medium containing chloramphenicol were inoculated directly from -80°C glycerol cell stock. Cells were grown in non-baffled-flasks at 37°C and 250 rpm.<br>  
The next day, main cultures in 75 mL 2xYT medium containing ampicillin were inoculated from overnight cultures to an OD520=0.5. Growth of each strain was observed with and without induction of the promoters regulation the ampicillin resistance gene. All cultures and measurements were conducted as duplicates.<br>  
The next day, main cultures in 75 mL 2xYT medium containing ampicillin were inoculated from overnight cultures to an OD520=0.5. Growth of each strain was observed with and without induction of the promoters regulation the ampicillin resistance gene. All cultures and measurements were conducted as duplicates.<br>  
-
For the induction of beta-lactamase (ampR) expression by pRhl und pLas autoinducers n-buturyl-homoserinlactone and n-oxododecanoyl-homoserinlactone were added respectively to a final concentration of 10 µmol/L. Cells were grown in non-baffled flasks at 37°C and 250 rpm. <br>  
+
For the induction of beta-lactamase (ampR) expression by P<sub>Rhl</sub> und P<sub>Las</sub> autoinducers N-buturyl-homoserine lactone and N-3-oxododecanoyl homoserine lactone were added respectively to a final concentration of 10 µmol/L. Cells were grown in non-baffled flasks at 37°C and 250 rpm. <br>  
-
Samples from each flask were taken at appropriate times depending on the growth phase of the cells. Optical density was measured at a wavelength of 520 nm in order to avoid/minimize absorption by the chromoproteins (see absorption spectra). Cells were left to grow until the stationary phase was reached.
+
Samples from each flask were taken at appropriate time points depending on the growth phase of the cells. Optical density was measured at a wavelength of 520 nm in order to avoid/minimize absorption by the chromoproteins (see absorption spectra). Cells were left to grow until the stationary phase was reached.
</p>
</p>
    
    
Line 173: Line 172:
<b>Electrocompetent cells</b><br>
<b>Electrocompetent cells</b><br>
-
A liquid culture of <i>E. Coli</i> was grown to an approximate OD<sub>520</sub> of 0.5 and subsequently incubated on ice for 30 min. Afterwards the cells were spun down for 15 min at 4000xg and 4 °C. The cell pellet was washed with 200 mL icecold sterile MiliQ and resuspended. Centrifugation and resuspension was repeated followed by another centrifugation at 4000xg and 4°C for 20 min and subsequent resuspension in 50 mL 10 % (v/v) icecold Glycerin. The cells were then spun down one last time for 10 min and 3220xg and resuspended in 400 µL icecold 10 % (v/v) Glycerin. The now electrocompetent cells were aliquoted, quick-frozen in liquid nitrogen and stored at -80°C.</p>
+
A liquid culture of <i>E. Coli</i> was grown to an approximate OD<sub>520</sub> of 0.5 and subsequently incubated on ice for 30 min. Afterwards the cells were spun down for 15 min at 4000xg and 4 °C. The cell pellet was washed with 200 mL icecold sterile MiliQ water and resuspended. Centrifugation and resuspension was repeated followed by another centrifugation at 4000xg and 4°C for 20 min and subsequent resuspension in 50 mL 10 % (v/v) icecold glycerol. The cells were then spun down one last time for 10 min and 3220xg and resuspended in 400 µL icecold 10 % (v/v) glycerol. The now electrocompetent cells were aliquoted, quick-frozen in liquid nitrogen and stored at -80°C.</p>
    
    
  </div>
  </div>
Line 188: Line 187:
Miniprep was done with Plasmid Miniprep Kit I by Peqlab (High Copy) following the manufacturer’s protocol.<br>  
Miniprep was done with Plasmid Miniprep Kit I by Peqlab (High Copy) following the manufacturer’s protocol.<br>  
First 2 mL of the cell suspension were spun down for 10 minutes (5000g).<br>  
First 2 mL of the cell suspension were spun down for 10 minutes (5000g).<br>  
-
For the alkaline lysis of the DNA the Pellet was resuspended in 250 μL Solution I (RNAseA). 250 μL of Solution II and 350 μL of solution III were consecutively added. As final step of the alkaline lysis the mixtures was centrifuged at 10.000 g for 10 minutes.<br>
+
For the alkaline lysis of the DNA the pellet was resuspended in 250 μL solution I (RNAseA). 250 μL of solution II and 350 μL of solution III were consecutively added. As final step of the alkaline lysis the mixtures was centrifuged at 10.000 g for 10 minutes.<br>
Afterwards the PerfectBindColumn was loaded with 750 μL lysate and then centrifuged for 1 minute (10000g). Subsequently the DNA was washed with 500 μL PW plasmid buffer and centrifuged again for 1 minute (10.000g). The DNA was the precipitated with the Ethanol containing wash buffer and separated by another centrifugation (1min, 10000g) and dried.<br>
Afterwards the PerfectBindColumn was loaded with 750 μL lysate and then centrifuged for 1 minute (10000g). Subsequently the DNA was washed with 500 μL PW plasmid buffer and centrifuged again for 1 minute (10.000g). The DNA was the precipitated with the Ethanol containing wash buffer and separated by another centrifugation (1min, 10000g) and dried.<br>
At last the DNA was eluted with 50 μL elution buffer, which was pre-heated to 60°C, and incubated for 5 min. The eluted DNA was separated from the matrix by a final centrifugation step (1 min, 5000g).  
At last the DNA was eluted with 50 μL elution buffer, which was pre-heated to 60°C, and incubated for 5 min. The eluted DNA was separated from the matrix by a final centrifugation step (1 min, 5000g).  
Line 222: Line 221:
     <h2><a href="#Gelslicepreparation">Gel Slice Preparation</a></h2>
     <h2><a href="#Gelslicepreparation">Gel Slice Preparation</a></h2>
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">
-
10 µL of 6x loading buffer were added to digested DNA und mixed. 55 µL of the sample were loaded onto a 1% agarose gel and separated via gel electrophoresis at 75 mA and xxxV for 20-40 min depending on the size of the DNA. For gel extraction the gel was visualized on an UV-lightsource and the appropriate bands were cut out with a razor blade. Gel purification was carried out using the Macherey-Nagel Nucleospin Gel and PCR Clean-up Kit.<br>
+
10 µL of 6x loading buffer were added to digested DNA und mixed. 55 µL of the sample were loaded onto a 1% agarose gel and separated via gel electrophoresis at 75 mA for 20-40 min depending on the size of the DNA. For gel extraction the gel was visualized on an UV-lightsource and the appropriate bands were cut using the x-tracta™ Gel Extractor tool (Promega). Gel purification was carried out using the Macherey-Nagel Nucleospin Gel and PCR Clean-up Kit.<br>
</p>
</p>
    
    
Line 231: Line 230:
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">
<b>Restriction digest</b><br>
<b>Restriction digest</b><br>
-
For restriction of vector and insert DNA ca. 1000 ng purified DNA, 5 µL 10x buffer and 1 µL of each of the two restriction enzymes were mixed in a 1.5 mL reaction tube. Water was added to a final volume of 50 µL. Restriction was carried out at 37 °C for 1 h. The reaction was stopped by inactivating the enzymes at 80 °C for 20 min.<br>
+
For restriction of vector and insert DNA approximately 1000 ng purified DNA, 5 µL 10x buffer and 1 µL of each of the two restriction enzymes were mixed in a 1.5 mL reaction tube. The amount of DNA used depended on the size of the desired Fragment. Water was added to a final volume of 50 µL. Restriction was carried out at 37 °C for 1 h. The reaction was stopped by inactivating the enzymes at 80 °C for 20 min.<br>
The buffer was chosen depending on the combination of restriction enzymes used:<br>  
The buffer was chosen depending on the combination of restriction enzymes used:<br>  
Line 249: Line 248:
<b>Transformation of chemocompetent cells via heatshock</b><br>
<b>Transformation of chemocompetent cells via heatshock</b><br>
-
One -80°C glycerol-stock of chemocompetend cells was thawed on ice. 10 µL of the ligated DNA was prepared in a 1.5 mL reaction tube. 50 µL of the cells were added and incubated for 20 min on ice. The mixture was then heat shocked at 42 °C for 1 min and cooled on ice for 2 min. 1 mL S.O.C.-medium was added and the cells were incubated for 1 h at 37 °C while shaken at 600 rpm.
+
One -80°C glycerol stock of chemocompetend cells was thawed on ice. 10 µL of the ligated DNA was prepared in a 1.5 mL reaction tube. 50 µL of the cells were added and incubated for 20 min on ice. The mixture was then heat shocked at 42 °C for 1 min and cooled on ice for 2 min. 1 mL SOC-medium was added and the cells were incubated for 1 h at 37 °C while shaken at 600 rpm.
-
100 µL of the cell suspension were plated on a 2xYT agar plate with the according antibiotic (Chloramphenicol or Ampicillin) and incubated at 37 °C over night.<br><br>
+
100 µL of the cell suspension were plated on a 2xYT agar plate with the according antibiotic (chloramphenicol or ampicillin) and incubated at 37 °C over night.<br><br>
</p>
</p>
Line 260: Line 259:
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">
-
One -80°C glycerol-stock of chemo competend cells was thawed on ice. 100 pg of purified DNA were prepared in an 1.5 mL reaction tube. 50 µL of the cells were added to the DNA and gently mixed. The cells were transferred into a precooled electroporation cuvette. Electroporation was carried out at 1.8 kV for 5.6 ms. The cells were resuspended in 1 mL S.O.C.-medium, transferred into a 2 mL reaction tube. The cells were incubated at 37 °C and shaken at 600 rpm for 1 h.<br>
+
One -80°C glycerol stock of chemocompetend cells was thawed on ice. 100 pg of purified DNA were prepared in an 1.5 mL reaction tube. 50 µL of the cells were added to the DNA and gently mixed. The cells were transferred into a precooled electroporation cuvette. Electroporation was carried out at 1.8 kV for 5.6 ms. The cells were resuspended in 1 mL S.O.C.-medium, transferred into a 2 mL reaction tube. The cells were incubated at 37 °C and shaken at 600 rpm for 1 h.<br>
-
100 µL of the cell suspension were plated on a 2xYT agar plate with antibiotic (Chloramphenicol or Ampicillin) and incubated at 37 °C overnight.
+
100 µL of the cell suspension were plated on a 2xYT agar plate with antibiotic (chloramphenicol or ampicillin) and incubated at 37 °C overnight.
</p>
</p>
Line 269: Line 268:
<div id="Proteinextraction" class="menuSection">
<div id="Proteinextraction" class="menuSection">
     <h2><a href="#Proteinextraction">Extraction of Chromoproteins</a></h2>
     <h2><a href="#Proteinextraction">Extraction of Chromoproteins</a></h2>
-
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">50 mL of overnight culture were pelleted for 10 min at 3220 g. The supernatant was discarded and the cells were resuspended in 4 mL of 1x Phosphate Buffered Saline (PBS).<br>
+
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">50 mL of overnight culture were pelleted for 10 min at 3220 g. The supernatant was discarded and the cells were resuspended in 4 mL of 1x phosphate buffered saline (PBS).<br>
Desintegration of the cells was performed by sonication (HD2200 Sonopuls, MS72 sonotrode, 50% performance, 2 x 2 min). The cell suspension was kept on ice during sonication and chilled for 5 min between the sonication cycles. The cell debris were pelleted for 5 min at 16,100 g. The supernatant containing the soluble chromoproteins was sterile filtered (0.2 µm) and stored at 4 °C.
Desintegration of the cells was performed by sonication (HD2200 Sonopuls, MS72 sonotrode, 50% performance, 2 x 2 min). The cell suspension was kept on ice during sonication and chilled for 5 min between the sonication cycles. The cell debris were pelleted for 5 min at 16,100 g. The supernatant containing the soluble chromoproteins was sterile filtered (0.2 µm) and stored at 4 °C.
</p>
</p>
Line 279: Line 278:
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">
<b>Absorption Spectra</b><br>
<b>Absorption Spectra</b><br>
-
In order to measure the absorption spectra of the different chromoproteins 100 mL 2xYT medium containing chloramphenicol was inoculated with E. coli XL1 including pSB1C3 with chromoprotein expression cassette and grown over night at 37°C and 250 rpm in non-baffled flask to limit oxygen transfer in order to enhance chromoprotein expression. The whole culture volume was centrifuged for 10 min at 6000 rpm and the supernatant discarded. Cells were resolved in 5 mL PBS and disrupted using ultrasonic technology (see protein purification). Subsequently the cells were centrifuged again at 6000 rpm the supernatant was transferred into a new tube and measured with the nanodrop in a range between 190-840 nm.<br><br>
+
In order to measure the absorption spectra of the different chromoproteins 100 mL 2xYT medium containing chloramphenicol was inoculated with <i>E. coli</i> XL1 Blue MRF' including pSB1C3 with chromoprotein expression cassette and grown over night at 37°C and 250 rpm in non-baffled flask to limit oxygen transfer in order to enhance chromoprotein expression. The whole culture volume was centrifuged for 10 min at 6000 rpm and the supernatant discarded. Cells were resolved in 5 mL PBS and disrupted using ultrasonic technology (see protein purification). Subsequently the cells were centrifuged again at 6000 rpm the supernatant was transferred into a new tube and measured with the nanodrop in a range between 190-840 nm.<br><br>
<b>Emmision Spectra</b><br>
<b>Emmision Spectra</b><br>
Line 288: Line 287:
<div id="Fluorescenceimaging" class="menuSection">
<div id="Fluorescenceimaging" class="menuSection">
     <h2><a href="#Fluorescenceimaging">Imaging of Cells producing Chromoproteins</a></h2>
     <h2><a href="#Fluorescenceimaging">Imaging of Cells producing Chromoproteins</a></h2>
-
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">Cells were grown in 100 ml 2xYT medium containing ampicillin and 10 µM n-buturyl-homoserinlactone (pRhl) or n-oxo-dodecanoyl-homosereinlactone (pLas) at 37°C and 250 rpm over night. The medium was inoculated directly from -80°C glycerol cell stock.  
+
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">Cells were grown in 100 ml 2xYT medium containing ampicillin and 10 µM N-buturyl homoserine lactone (P<sub>Rhl</sub>) or N-3-oxododecanoyl homoserine lactone (P<sub>Las</sub>) at 37°C and 250 rpm over night. The medium was inoculated directly from -80°C glycerol cell stock.  
Cells were diluted with fresh 2xYT medium containing ampicillin and suspension was placed between to agar layers containing ampicllin as well.<br>  
Cells were diluted with fresh 2xYT medium containing ampicillin and suspension was placed between to agar layers containing ampicllin as well.<br>  
Cell growth and division were observed for 3 hours by exciting chromoproteins every five minutes at specific wavelength (see absorption spectra) using YFP und mCherry filters.  
Cell growth and division were observed for 3 hours by exciting chromoproteins every five minutes at specific wavelength (see absorption spectra) using YFP und mCherry filters.  
Line 299: Line 298:
<div id="Inducerdetection" class="menuSection">
<div id="Inducerdetection" class="menuSection">
     <h2><a href="#Inducerdetection">Detection of Autoinducers</a></h2>
     <h2><a href="#Inducerdetection">Detection of Autoinducers</a></h2>
-
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">For the production of autoinducers liquid cultures (2xYT medium containing Chloramphenicol) of <i>E. Coli</i> JM109 bearing the BioBrick BBa_K1073034 and <i>E. Coli</i> TOP10F’ bearing the construct BBa_K1073035 were inoculated and incubated overnight. Once the producing cultures reached an OD520 of about 11, cells were harvested by centrifugation for 10 min at 4000 rpm. The biomass was discarded and the supernatant sterilized by filtration. Subsequently the supernatant was diluted 1:2 with sterile 2x YT medium containing Ampicillin.<br>  
+
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">For the production of autoinducers liquid cultures (2xYT medium containing Chloramphenicol) of <i>E. Coli</i> JM109 bearing the BioBrick BBa_K1073034 and <i>E. Coli</i> TOP10F’ bearing the construct BBa_K1073035 were inoculated and incubated overnight. Once the producing cultures reached an OD520 of about 11, cells were harvested by centrifugation for 10 min at 4000 rpm. The biomass was discarded and the supernatant sterilized by filtration. Subsequently the supernatant was diluted 1:2 with sterile 2x YT medium containing ampicillin.<br>  
For the detection of the autoinducers cultures of the reporter strains <i>E. coli</i> JM109 pSB406 for  N-(butyryl)-homoserine lactone detection  and <i>E. coli</i> JM109 pSB1075 for N-(3-oxododecanoyl)-homoserine lactone detection were grown overnight on 2x YT medium supplemented with Ampicillin. The next day the cultures were diluted 1:15 with the above described supernatant solution and incubated for 3 h at 37 °C and 300 rpm in a 96 well plate.<br>
For the detection of the autoinducers cultures of the reporter strains <i>E. coli</i> JM109 pSB406 for  N-(butyryl)-homoserine lactone detection  and <i>E. coli</i> JM109 pSB1075 for N-(3-oxododecanoyl)-homoserine lactone detection were grown overnight on 2x YT medium supplemented with Ampicillin. The next day the cultures were diluted 1:15 with the above described supernatant solution and incubated for 3 h at 37 °C and 300 rpm in a 96 well plate.<br>
Line 312: Line 311:
<div id="Agardiffusiontest" class="menuSection">
<div id="Agardiffusiontest" class="menuSection">
     <h2><a href="#Agardiffusiontest">Agar Diffusion Test</a></h2>
     <h2><a href="#Agardiffusiontest">Agar Diffusion Test</a></h2>
-
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">Overnight cultures of <i>E. Coli</i> JM109 carrying BBa_K1073034 and <i>E. Coli</i> TOP10F’ carrying BBa_K1073035 were grown on 2x YT medium containing Ampicillin and the respective autoinducer. The next day the cells were harvested by centrifugation at 4000 rpm for 10 min and subsequently resuspended in fresh 2xYT medium. The obtained cultures were then spread out on Ampicillin containing agar plates. A filter paper impregnated with the respective synthetic autoinducer solution was laid on top of the freshly spread agar plate. Afterwards the plates were incubated at 37 °C for 3-4 h and pictures were taken to document the circles of bacterial growth around the filter.<br>
+
     <p><p style=" margin-left:5px; margin-right:5px; margin-bottom:5px;">Overnight cultures of <i>E. Coli</i> JM109 carrying BBa_K1073034 and <i>E. Coli</i> TOP10F’ carrying BBa_K1073035 were grown on 2xYT medium containing ampicillin and the respective autoinducer. The next day the cells were harvested by centrifugation at 4000 rpm for 10 min and subsequently resuspended in fresh 2xYT medium. The obtained cultures were then spread out on ampicillin containing agar plates. A filter paper impregnated with the respective synthetic autoinducer solution was placed on top of the freshly spread agar plate. Afterwards the plates were incubated at 37 °C for 3-4 h and pictures were taken to document the circles of bacterial growth around the filter.<br>
This experiment was also conducted with the supernatant of the respectively other strain as the two strains cross induce each other.
This experiment was also conducted with the supernatant of the respectively other strain as the two strains cross induce each other.
</p>
</p>

Revision as of 20:31, 3 October 2013

Protocols

linie rot 8pix hoch

In this section you will find detailed protocols of experimental procedures.

Our sponsors

linie rot 8pix hoch