Team:Braunschweig/Notebook

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     <h2><a href="#Week3">Week 3: June 2 - June 8, 2013</a></h2>
     <h2><a href="#Week3">Week 3: June 2 - June 8, 2013</a></h2>
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     <p style=" margin-left:5px; margin-right:5px;">
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<img alt="Transformation" src="https://static.igem.org/mediawiki/2013/f/f5/Braunschweig_Lab_Week3_Transformation.jpg" width="250" align="left" vspace="0" hspace="20"/>
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<img alt="Transformation" src="https://static.igem.org/mediawiki/2013/f/f5/Braunschweig_Lab_Week3_Transformation.jpg" width="250" align="left" vspace="0" hspace="20"style="margin-right:5px"/>
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This week we successfully cloned and transformed some of our first combination Bricks. We also managed to obtain some BioBricks that could not be transformed via Phusion-PCR directly from resuspended DNA.</p>
This week we successfully cloned and transformed some of our first combination Bricks. We also managed to obtain some BioBricks that could not be transformed via Phusion-PCR directly from resuspended DNA.</p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Roman, Laura</b><br>
<b>Investigators: Roman, Laura</b><br>
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Since the transformations of the BioBricks B0010, C0060, C0061, C0062, C0070, J23100 and J23106 were not successful, These Bricks were amplified via Phusion-PCR directly from the resuspended DNA from the distribution kit instead.<br>
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Since the transformations of the BioBricks B0010, C0060, C0061, C0062, C0070, J23100 and J23106 were not successful, these Bricks were amplified via Phusion-PCR directly from the resuspended DNA from the distribution kit instead.<br>
We prepared 5 mL liquid cultures of Bricks B0012, B0032, B1009, C0060, C0061, C0062, C0070, C0071, C0078, C0079, E0240, E0430, J06702, R0062, R0071 in 2xYT medium containing respective antibiotics in order to miniprep the plasmid DNA tomorrow.</p>
We prepared 5 mL liquid cultures of Bricks B0012, B0032, B1009, C0060, C0061, C0062, C0070, C0071, C0078, C0079, E0240, E0430, J06702, R0062, R0071 in 2xYT medium containing respective antibiotics in order to miniprep the plasmid DNA tomorrow.</p>
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Today we received the NEB iGEM Support Kit. This will keep the labwork rolling ;-)<br>
Today we received the NEB iGEM Support Kit. This will keep the labwork rolling ;-)<br>
Yesterday’s colony PCR(E0420 and C0061+B0015) was analyzed on a 1% agarose gel.The amplified fragment for E0420 matched the expected fragment size of 1192 bp. The PCR for C0061+B0015 failed. A faint PCR product of 6-7kb could be detected for clone 2 indicating (again) an additional oligonucleotide sequence 5’ of the BioBrick. This additional DNA sequence may have up to 6kb. We isolated the plasmid DNA from all clones of E0420 and C0061 + B0015 with a miniprep kit following the manufacturer’s instructions to send them to GATC.<br>
Yesterday’s colony PCR(E0420 and C0061+B0015) was analyzed on a 1% agarose gel.The amplified fragment for E0420 matched the expected fragment size of 1192 bp. The PCR for C0061+B0015 failed. A faint PCR product of 6-7kb could be detected for clone 2 indicating (again) an additional oligonucleotide sequence 5’ of the BioBrick. This additional DNA sequence may have up to 6kb. We isolated the plasmid DNA from all clones of E0420 and C0061 + B0015 with a miniprep kit following the manufacturer’s instructions to send them to GATC.<br>
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In order to clone the inducible promoters P<sub>lux</sub> (R0062), P<sub>rhl</sub> (R0071) and P<sub>las</sub> (K091117) 5’ of the RBS (B0032), we digested them with EcoRI and SpeI. At the same time the lactonase (C0060) and lactonase + TT were digested with XbaI and PstI to clone them 3’ of a RBS. The autoinducer synthases + TT (C0078+B0015, C0070+B0015, C0061+B0015) were digested with XbaI and PstI for cloning. Also we did a test restriction of C0061 since the DNA sequence indicated it contained an additional nucleotide sequence.<br><br><br>
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In order to clone the inducible promoters Plux (R0062), Prhl (R0071) and Plas (K091117) 5’ of the RBS (B0032), we digested them with EcoRI and SpeI. At the same time the lactonase (C0060) and lactonase + TT were digested with XbaI and PstI to clone them 3’ of a RBS. The autoinducer synthases + TT (C0078+B0015, C0070+B0015, C0061+B0015) were digested with XbaI and PstI for cloning. Also we did a test restriction of C0061 since the DNA sequence indicated it contained an additional nucleotide sequence.<br><br><br>
<img alt="June13" src="https://static.igem.org/mediawiki/2013/3/30/Braunschweig_Lab_Journal_June_13_2.png" width="200" align="right" vspace="0" hspace="10"/>
<img alt="June13" src="https://static.igem.org/mediawiki/2013/3/30/Braunschweig_Lab_Journal_June_13_2.png" width="200" align="right" vspace="0" hspace="10"/>
The expected fragments of the promoters are 82, 80 and 153 bp.  We were not able to detect the fragments on the agarose gel. The restriction digest of C0060, C0078+B0015 and C0070+B0015 was not complete. However, the fragments matched the expected sizes, so they were extracted from the agarose gel.<br>
The expected fragments of the promoters are 82, 80 and 153 bp.  We were not able to detect the fragments on the agarose gel. The restriction digest of C0060, C0078+B0015 and C0070+B0015 was not complete. However, the fragments matched the expected sizes, so they were extracted from the agarose gel.<br>
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<p><img alt="Chromoprotein arrival" src="https://static.igem.org/mediawiki/2013/2/2e/Braunschweig_Lab_Week7_Chromoproteina_arrival.jpg" height="150px" align="left" vspace="0" hspace="10" style="margin-right:5px"/>
<p><img alt="Chromoprotein arrival" src="https://static.igem.org/mediawiki/2013/2/2e/Braunschweig_Lab_Week7_Chromoproteina_arrival.jpg" height="150px" align="left" vspace="0" hspace="10" style="margin-right:5px"/>
Even if we switched to our new strategy we still kept working on the fluorescence markers. Therefore we digested the BioBricks E0420 (eCFP), E0430 (YFP) and J06702 (mCherry) as well as our promotor-RBS-LasR and promotor-RBS-RhlR-constructs. Still lacking the construct containing LuxR we decided to proceed with the promotor-RBS-construct instead to ligate it to the YFP-Brick. Gel extraction was performed for the inserts and DNA purification for the vector parts. Inserts and bricks were ligated overnight using T4 DNA ligase (NEB) at 16°C.<br><br><br>
Even if we switched to our new strategy we still kept working on the fluorescence markers. Therefore we digested the BioBricks E0420 (eCFP), E0430 (YFP) and J06702 (mCherry) as well as our promotor-RBS-LasR and promotor-RBS-RhlR-constructs. Still lacking the construct containing LuxR we decided to proceed with the promotor-RBS-construct instead to ligate it to the YFP-Brick. Gel extraction was performed for the inserts and DNA purification for the vector parts. Inserts and bricks were ligated overnight using T4 DNA ligase (NEB) at 16°C.<br><br><br>
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Since we now have our first constructs containing the inducible promotors as well as the the ampicillin resistence gen we started our first leakiness experiments. All constructs (P<sub>las</sub>, P<sub>rhl</sub>, P<sub>lux</sub> in combination with the RBS and <i>ampR</i>) were cultivated overnight in 2xYT medium containing various ampicillin concentrations.</p>
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Since we now have our first constructs containing the inducible promotors as well as the the ampicillin resistence gen we started our first leakiness experiments. All constructs (Plas, Prhl, Plux in combination with the RBS and <i>ampR</i>) were cultivated overnight in 2xYT medium containing various ampicillin concentrations.</p>
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<b>Investigators: Laura, Kerstin </b><br>
<b>Investigators: Laura, Kerstin </b><br>
Before we started working on our own chromoprotein-constructs, we screened for the optimum cultivation conditions. We cultivated <i>E. coli</i> XL1 Blue MRF' containing a new construct from Uppsala iGEM encoding the device J23110-B0034-aeBlue. We tested expression of the blue chromoprotein at different temperatures, oxygen supply and rpm. These experiments led to the conclusion that low oxygen supply and a temperature of 37°C result in higher expression rates.<br><br>
Before we started working on our own chromoprotein-constructs, we screened for the optimum cultivation conditions. We cultivated <i>E. coli</i> XL1 Blue MRF' containing a new construct from Uppsala iGEM encoding the device J23110-B0034-aeBlue. We tested expression of the blue chromoprotein at different temperatures, oxygen supply and rpm. These experiments led to the conclusion that low oxygen supply and a temperature of 37°C result in higher expression rates.<br><br>
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The evaluation of the leakiness of the inducible promotors showed that only the P<sub>rhl</sub> is not leaky. P<sub>lux</sub> as well as P<sub>las</sub> were leaky and showed growth of <i>E. coli</i> XL1 Blue MRF' at all tested ampicillin concentrations. Therefore we repeated the experiment using higher ampicillin conentrations.<br><br>
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The evaluation of the leakiness of the inducible promotors showed that only the Prhl is not leaky. Plux as well as Plas were leaky and showed growth of <i>E. coli</i> XL1 Blue MRF' at all tested ampicillin concentrations. Therefore we repeated the experiment using higher ampicillin conentrations.<br><br>
The bricks containing the fluorescencw markers ligated yesterday were transformed in <i>E. coli</i> XL1 Blue MRF' by heatshock.<br><br>
The bricks containing the fluorescencw markers ligated yesterday were transformed in <i>E. coli</i> XL1 Blue MRF' by heatshock.<br><br>
<img alt="July3" src="https://static.igem.org/mediawiki/2013/f/fa/Braunschweig_Lab_Journal_July_3.png" width="200" align="right" vspace="0" hspace="10"/>Colony PCR of the constructs containing lactonase and LuxI which were transformed yesterday was performed. Since all screened colonies showed religated vectors we decided to try an alternative restriction strategy to combine the BioBricks by using the endonuclease NcoI.<br>   
<img alt="July3" src="https://static.igem.org/mediawiki/2013/f/fa/Braunschweig_Lab_Journal_July_3.png" width="200" align="right" vspace="0" hspace="10"/>Colony PCR of the constructs containing lactonase and LuxI which were transformed yesterday was performed. Since all screened colonies showed religated vectors we decided to try an alternative restriction strategy to combine the BioBricks by using the endonuclease NcoI.<br>   
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The digested chromoprotein DNA was ligated with the RBS and the promotor-RBS construct at room temperature for 30 minutes. The ligated DNA was transformed in <i>E. coli</i> XL1 Blue MRF' by heatshock and plated on agar plates. We cannot wait to see colorful colonies on Sunday :-)<br>
The digested chromoprotein DNA was ligated with the RBS and the promotor-RBS construct at room temperature for 30 minutes. The ligated DNA was transformed in <i>E. coli</i> XL1 Blue MRF' by heatshock and plated on agar plates. We cannot wait to see colorful colonies on Sunday :-)<br>
Since we almost ran out off our competent cells it was time for new ones. New compentent cells were made and transformation efficiency was tested.<br>
Since we almost ran out off our competent cells it was time for new ones. New compentent cells were made and transformation efficiency was tested.<br>
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A new idea to cope with the leakiness of the P<sub>las</sub> and P<sub>lux</sub> was to try a different antibiotic which cannot be metabolised. We used carbenicillin instead of ampicillin at different concentrations in liquid culture with 2xYT medium. Unfortunatly no effect could be observed on the leakyness of both inducible promotors.</p>   
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A new idea to cope with the leakiness of the Plas and Prhl was to try a different antibiotic which cannot be metabolised. We used carbenicillin instead of ampicillin at different concentrations in liquid culture with 2xYT medium. Unfortunatly no effect could be observed on the leakyness of both inducible promotors.</p>   
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   </div>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Laura, Kerstin, Kevin</b><br>
<b>Investigators: Laura, Kerstin, Kevin</b><br>
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The results of the leakiness experiment: The growth of cells equipped with the P<sub>Las</sub> inducible ampicillin resistance cassette, probably a double clone, was firstly limited at ampicillin concentration of 400µg/ml. The P<sub>Las</sub> inducible ampicillin resistance cassette and the positive control grew at all tested concentrations of ampicillin (100µg/ml-1 mg/ml).  
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The results of the leakiness experiment: The growth of cells equipped with the Plas inducible ampicillin resistance cassette, probably a double clone, was firstly limited at ampicillin concentration of 400µg/ml. The Plas inducible ampicillin resistance cassette and the positive control grew at all tested concentrations of ampicillin (100µg/ml-1 mg/ml).  
To show that the transformation of our chromoprotein expression cassettes with and without strong constitutive promoters was successful, colony PCR was performed.
To show that the transformation of our chromoprotein expression cassettes with and without strong constitutive promoters was successful, colony PCR was performed.
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The constitutive RhlR and LasR transcription factor expression cassettes were digested with  appropriate restriction enzymes and desired fragments were extracted from gel after gelelectrophoresis. Lastly, the DNA was purified. Transformation of the PLas inducible ampicillin resistance cassette was performed. However, after prepping the DNA and sending it for sequencing the sequence revealed that this construct was a double clone.
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The constitutive RhlR and LasR transcription factor expression cassettes were digested with  appropriate restriction enzymes and desired fragments were extracted from gel after gelelectrophoresis. Lastly, the DNA was purified. Transformation of the Plas inducible ampicillin resistance cassette was performed. However, after prepping the DNA and sending it for sequencing the sequence revealed that this construct was a double clone.
The earlier mentioned transcription factor expression cassettes for RhlR and LasR were then ligated and incubated overnight. Also, 2xYT liquid cultures of all chromoprotein expression cassettes were prepared and incubated overnight.</p>  
The earlier mentioned transcription factor expression cassettes for RhlR and LasR were then ligated and incubated overnight. Also, 2xYT liquid cultures of all chromoprotein expression cassettes were prepared and incubated overnight.</p>  
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Today, our aim was to combine the chromoprotein expression cassettes with the double terminator (B0015). Thus, the chromoprotein expression cassettes were digested, a gel electrophoresis was run and insert fragments were extracted from gel while the vector part was dephosphorylated. Afterwards purification of vector and insert parts was performed and the DNA was ligated according to our protocol. Subsequently, the ligated DNA was transformed into <i>E. coli</i> XL1 Blue MRF' by heat shock.  
Today, our aim was to combine the chromoprotein expression cassettes with the double terminator (B0015). Thus, the chromoprotein expression cassettes were digested, a gel electrophoresis was run and insert fragments were extracted from gel while the vector part was dephosphorylated. Afterwards purification of vector and insert parts was performed and the DNA was ligated according to our protocol. Subsequently, the ligated DNA was transformed into <i>E. coli</i> XL1 Blue MRF' by heat shock.  
Yesterday‘s test for leakiness under different conditions showed that the use of LB Medium wouldn’t solve the problem of the leaky promoters since liquid cultures showed high cell density for all ampicillin concentrations. We still need to find a solution for this problem in order to get our concept to work.  
Yesterday‘s test for leakiness under different conditions showed that the use of LB Medium wouldn’t solve the problem of the leaky promoters since liquid cultures showed high cell density for all ampicillin concentrations. We still need to find a solution for this problem in order to get our concept to work.  
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Furthermore, we performed new colony PCRs and agarose ge of the transcription factor LuxR, the combined Rhl-autoinducer synthase RhlI and aeBlue expression cassette as well as the P<sub>Las</sub> induced ampicillin resistance cassette from colonies on agar plates.<p>  
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Furthermore, we performed new colony PCRs and agarose ge of the transcription factor LuxR, the combined Rhl-autoinducer synthase RhlI and aeBlue expression cassette as well as the Plas induced ampicillin resistance cassette from colonies on agar plates.<p>  
   </div>
   </div>
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     <h2><a href="#Week10">Week 10: July 21 - July 27, 2013</a></h2>
     <h2><a href="#Week10">Week 10: July 21 - July 27, 2013</a></h2>
     <p><p style=" margin-left:5px; margin-right:5px;">
     <p><p style=" margin-left:5px; margin-right:5px;">
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This week we managed to construct our N-buturyl-HSL (Rhl system) inducible ampicillin resistance cassette (R0071-B0032-AmpR-B0015-J23100-B0032-C0071) and our N-3-oxododecanoyl-HSL (Las system) inducible ampicillin resistance cassette (K091117-B0032-AmpR-B0015-J23100-B0032-C0079). Furthermore, the constructs of the amilGFP cassette (J23100-B0032-K592010-B0015), the N-3-oxododecanoyl-HSL synthase LasI and eforRed cassette (B0032-C0076-B0015-J23100-B0032-K592012-B0015), the N-buturyl-HSL synthase RhlI and aeBlue cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015) and terminator-modified N-buturyl-HSL (Rhl system) inducible Amp-resistance cassette (B0015-R0071-B0032-AmpR-B0015-J23100-B0032-C0071) are nearly finished.
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This week we managed to construct our N-butyryl-HSL (Rhl system) inducible ampicillin resistance cassette (R0071-B0032-AmpR-B0015-J23100-B0032-C0071) and our N-3-oxododecanoyl-HSL (Las system) inducible ampicillin resistance cassette (K091117-B0032-AmpR-B0015-J23100-B0032-C0079). Furthermore, the constructs of the amilGFP cassette (J23100-B0032-K592010-B0015), the N-3-oxododecanoyl-HSL synthase LasI and eforRed cassette (B0032-C0076-B0015-J23100-B0032-K592012-B0015), the N-butyryl-HSL synthase RhlI and aeBlue cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015) and terminator-modified N-butyryl-HSL (Rhl system) inducible Amp-resistance cassette (B0015-R0071-B0032-AmpR-B0015-J23100-B0032-C0071) are nearly finished.
Additionally we started our first test runs with the new chromoproteins.</p>
Additionally we started our first test runs with the new chromoproteins.</p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Kevin, Kerstin, Laura</b><br>
<b>Investigators: Kevin, Kerstin, Laura</b><br>
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We digested the N-buturyl-HSL/RhlR induced ampicillin resistance cassette (R0071-B0032-<i>ampR</i>) , the Rhl transcription factor (RhlR) cassette (B0015-J23100)-B0032-C0071), the N-3-oxododecanoyl-HSL/LasR induced ampicillin resistance cassette (K091117-B0032-<i>ampR</i>) and the Las-transcription factor (LasR) cassette (B0015-J23100-B0032-C0079) to ligate them to new constructs. The N-buturyl-HSL/RhlR inducible ampicillin resistance cassette (R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) is supposed to be combined with the RhlR cassettte while the the N-3-oxododecanoyl-HSL/LasR inducible ampicillin resistance cassette (K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) is intended to be combined with the LasR expression cassette. We successfully performed a gel extraction of the RhlR cassette (B0015-J23100-B0032-C0071) and the LasR cassette (B0015-J23100-B0032-C0079). Additionally we measured the absorption spectra emission of our newly received chromoproteins.
+
We digested the N-butyryl-HSL/RhlR induced ampicillin resistance cassette (R0071-B0032-<i>ampR</i>) , the Rhl transcription factor (RhlR) cassette (B0015-J23100)-B0032-C0071), the N-3-oxododecanoyl-HSL/LasR induced ampicillin resistance cassette (K091117-B0032-<i>ampR</i>) and the Las-transcription factor (LasR) cassette (B0015-J23100-B0032-C0079) to ligate them to new constructs. The N-butyryl-HSL/RhlR inducible ampicillin resistance cassette (R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) is supposed to be combined with the RhlR cassettte while the the N-3-oxododecanoyl-HSL/LasR inducible ampicillin resistance cassette (K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) is intended to be combined with the LasR expression cassette. We successfully performed a gel extraction of the RhlR cassette (B0015-J23100-B0032-C0071) and the LasR cassette (B0015-J23100-B0032-C0079). Additionally we measured the absorption spectra emission of our newly received chromoproteins.
</p>
</p>
<p><img alt="Absorption amilGFP" src="https://static.igem.org/mediawiki/2013/e/e5/Braunschweig_Absorption-amilGFP.jpg" width="280px"  vspace="10" hspace="10"/>
<p><img alt="Absorption amilGFP" src="https://static.igem.org/mediawiki/2013/e/e5/Braunschweig_Absorption-amilGFP.jpg" width="280px"  vspace="10" hspace="10"/>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Kevin</b><br>
<b>Investigators: Kevin</b><br>
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Today we transformed yesterday’s ligations of the N-buturyl-HSL/RhlR inducicle ampicillin resistance cassette (R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) and the N-3-oxododecanoyl-HSL/LasR inducible ampicillin resistance cassette (K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079).
+
Today we transformed yesterday’s ligations of the N-butyryl-HSL/RhlR inducicle ampicillin resistance cassette (R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) and the N-3-oxododecanoyl-HSL/LasR inducible ampicillin resistance cassette (K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079).
Furthermore, we inoculated overnight cultures of the <i>E. coli</i> XL1 Blue MRF' expressing the chromoproteins for further spectrum measurements. Cultures were grown in 2xYT containing chloramphenicol at 37°C and 250 rpm overnight.</p>
Furthermore, we inoculated overnight cultures of the <i>E. coli</i> XL1 Blue MRF' expressing the chromoproteins for further spectrum measurements. Cultures were grown in 2xYT containing chloramphenicol at 37°C and 250 rpm overnight.</p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Kevin, Kerstin</b><br>
<b>Investigators: Kevin, Kerstin</b><br>
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A colony PCR of the successfully transformed N-buturyl-HSL/RhlR inducible ampicillin resistance cassette (R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) and N-3-oxododecanoyl-HSL/LasR inducible ampicillin resistance cassette (K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) was performed. We also extracted the chromoproteins by performing a cell disruption and measured their emission spectra.</p>
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A colony PCR of the successfully transformed N-butyryl-HSL/RhlR inducible ampicillin resistance cassette (R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) and N-3-oxododecanoyl-HSL/LasR inducible ampicillin resistance cassette (K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) was performed. We also extracted the chromoproteins by performing a cell disruption and measured their emission spectra.</p>
<p><img alt="Emission amilGFP" src="https://static.igem.org/mediawiki/parts/7/73/BS2013Emission_amilGFP.jpg" width="280px"  vspace="10" hspace="10"/>
<p><img alt="Emission amilGFP" src="https://static.igem.org/mediawiki/parts/7/73/BS2013Emission_amilGFP.jpg" width="280px"  vspace="10" hspace="10"/>
<img alt="Emission aeBlue" src="https://static.igem.org/mediawiki/parts/f/f6/BS2013Emission_aeBlue.jpg" width="280px"  vspace="10" hspace="10"/>
<img alt="Emission aeBlue" src="https://static.igem.org/mediawiki/parts/f/f6/BS2013Emission_aeBlue.jpg" width="280px"  vspace="10" hspace="10"/>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Laura, Kerstin</b><br>
<b>Investigators: Laura, Kerstin</b><br>
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We confirmed that our N-buturyl-HSL/RhlR inducicle ampicillin resistance cassette and RhlR cassette (R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) as well as our N-3-oxododecanoyl-HSL/LasR inducible ampicillin resistance cassette and LasR Cassette (K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) were correctly ligated by gel electrophoresis. Both contructs showed PCR fragments of expected size. In order to obtain material for sequencing an overnight culture of 2xYT containing chloramphenicol was inoculated with clones bearing the right contructs.</p>
+
We confirmed that our N-butyryl-HSL/RhlR inducicle ampicillin resistance cassette and RhlR cassette (R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) as well as our N-3-oxododecanoyl-HSL/LasR inducible ampicillin resistance cassette and LasR Cassette (K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) were correctly ligated by gel electrophoresis. Both contructs showed PCR fragments of expected size. In order to obtain material for sequencing an overnight culture of 2xYT containing chloramphenicol was inoculated with clones bearing the right contructs.</p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Laura, Kerstin</b><br>
<b>Investigators: Laura, Kerstin</b><br>
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The N-buturyl-HSL/RhlR inducicle ampicillin resistance cassette (R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) and the N-3-oxododecanoyl-HSL/LasR inducible ampicillin resistance cassette (K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) were minipreped for sequencing.<br>
+
The N-butyryl-HSL/RhlR inducicle ampicillin resistance cassette (R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) and the N-3-oxododecanoyl-HSL/LasR inducible ampicillin resistance cassette (K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) were minipreped for sequencing.<br>
-
The constructs of the amilGFP cassette (J23100-B0032-K592010-B0015), the N-3-oxododecanoyl-HSL synthase LasI and eforRed cassette (B0032-C0076-B0015-J23100-B0032-K592012-B0015), the N-buturyl-HSL synthase RhlI and aeBlue cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015) and the terminator-modified N-buturyl-HSL/RhlR inducible ampicillin resistance cassette (B0015-R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) were digested, ligated and transformed.<br>
+
The constructs of the amilGFP cassette (J23100-B0032-K592010-B0015), the N-3-oxododecanoyl-HSL synthase LasI and eforRed cassette (B0032-C0076-B0015-J23100-B0032-K592012-B0015), the N-butyryl-HSL synthase RhlI and aeBlue cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015) and the terminator-modified N-butyryl-HSL/RhlR inducible ampicillin resistance cassette (B0015-R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) were digested, ligated and transformed.<br>
As we were not able to transform the pSB1C3 plasmid of BBa_B0015, we performed a Q5 PCR of the part. The fragment was extracted from a gel and purified for further use.</p>
As we were not able to transform the pSB1C3 plasmid of BBa_B0015, we performed a Q5 PCR of the part. The fragment was extracted from a gel and purified for further use.</p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Kerstin, Laura</b><br>
<b>Investigators: Kerstin, Laura</b><br>
-
<img alt="July 29" src="https://static.igem.org/mediawiki/2013/f/f1/Braunschweig_Lab_Journal_July_29.png" width="450"  vspace="20" align="right"/>We transformed our inducible ampicillin resistance into a new <i>E. coli</i> strain (JM109) in order to test whether the promotor being leaky is strain specific.<br>  
+
<img alt="July 29" src="https://static.igem.org/mediawiki/2013/f/f1/Braunschweig_Lab_Journal_July_29.png" width="450"  vspace="20" align="right" style="margin-left:5px"/>We transformed our inducible ampicillin resistance into a new <i>E. coli</i> strain (JM109) in order to test whether the promotor being leaky is strain specific.<br>  
-
We performed a colony PCR to test the result of our latest cloning experiments for the amilGFP cassette (J23100-B0032-K592010-B0015), the inducible ampicillin resistance and LasR transcription factor cassette (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079), the N-3-oxododecanoyl-HSL synthase with eforRed reporter cassette (B0032-C0076-B0015-J23100-B0032-K592012) and the N-buturyl-HSL synthase with aeBlue reporter cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015).<br>
+
We performed a colony PCR to test the result of our latest cloning experiments for the amilGFP cassette (J23100-B0032-K592010-B0015), the inducible ampicillin resistance and LasR transcription factor cassette (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079), the N-3-oxododecanoyl-HSL synthase with eforRed reporter cassette (B0032-C0076-B0015-J23100-B0032-K592012) and the N-butyryl-HSL synthase with aeBlue reporter cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015).<br>
-
The amilGFP cassette (J23100-B0032-K592010-B0015), the N-3-oxododecanoyl-HSL synthase LasI with eforRed reporter cassette (B0032-C0076-B0015-J23100-B0032-K592012) and the N-buturyl-HSL synthase RhlI with aeBlue reporter cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015) showed the expected bands on the gel and were prepped. The inducible ampicillin resistance and LasR transcription factor cassette (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) was prepped as well despite a second colony PCR showing no positive results.<br>
+
The amilGFP cassette (J23100-B0032-K592010-B0015), the N-3-oxododecanoyl-HSL synthase LasI with eforRed reporter cassette (B0032-C0076-B0015-J23100-B0032-K592012) and the N-butyryl-HSL synthase RhlI with aeBlue reporter cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015) showed the expected bands on the gel and were prepped. The inducible ampicillin resistance and LasR transcription factor cassette (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) was prepped as well despite a second colony PCR showing no positive results.<br>
-
We also tested the inducibility of our final P<sub>Rhl</sub> construct in 2xYT liquid culture with ampicillin overnight, which showed normal growth without being induced, indicating that the promotor is still leaky.<br></p>
+
We also tested the inducibility of our final Prhl construct in 2xYT liquid culture with ampicillin overnight, which showed normal growth without being induced, indicating that the promotor is still leaky.<br></p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Roman, Kevin, Anna</b><br>
<b>Investigators: Roman, Kevin, Anna</b><br>
-
We started a restriction digest of several bricks to construct three new parts: The Inducible promoter with Rhl expression cassette (R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) and the LasI with eforRed reporter cassette (B0032-C0076-B0015-J23100-B0032-K592012) were combined to our first complete construct (fianl P<sub>Rhl</sub> construct).<br>  
+
We started a restriction digest of several bricks to construct three new parts: The Inducible promoter with Rhl expression cassette (R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) and the LasI with eforRed reporter cassette (B0032-C0076-B0015-J23100-B0032-K592012) were combined to our first complete construct (fianl Prhl construct).<br>  
The RhlI with amilGFP reporter cassette (B0032-C0070-B0015-J23100-B0032-K592010-B0015) was derived from the RhlI expression cassette (B0032-C0070-B0015) and the amilGFP cassette (J23100-B0032-K592010-B0015) as the blue chromoprotein was not usable for fluorescence detection.<br>
The RhlI with amilGFP reporter cassette (B0032-C0070-B0015-J23100-B0032-K592010-B0015) was derived from the RhlI expression cassette (B0032-C0070-B0015) and the amilGFP cassette (J23100-B0032-K592010-B0015) as the blue chromoprotein was not usable for fluorescence detection.<br>
-
The second final construct (BBa_K1073034), which is N-3-oxododecanoyl/LasR inducible, was constructed from the inducible ampicillin resistance and LasR transcription factor cassette (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) and the N-buturyl-HSL synthase RhlI with aeBlue reporter cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015).<br>
+
The second final construct (BBa_K1073034), which is N-3-oxododecanoyl/LasR inducible, was constructed from the inducible ampicillin resistance and LasR transcription factor cassette (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) and the N-butyryl-HSL synthase RhlI with aeBlue reporter cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015).<br>
Parts E0420 and J23100 were combined as another alternative for fluorescence experiments.<br>
Parts E0420 and J23100 were combined as another alternative for fluorescence experiments.<br>
We tested the results of yesterday's cloning experiments via colony PCR. C0062 and C0061-B0015 did not show bands of expected size.</p>  
We tested the results of yesterday's cloning experiments via colony PCR. C0062 and C0061-B0015 did not show bands of expected size.</p>  
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Kevin, Anna, Melanie</b><br>
<b>Investigators: Kevin, Anna, Melanie</b><br>
-
<img alt="August 2" src="https://static.igem.org/mediawiki/2013/c/cb/Braunschweig_Lab_Journal_August_2.png" width="250"  vspace="20" align="right"/>In order to test the success of the last days’ cloning experiment we conducted a colony PCR. Unfortunately all clones of both our supposed final P<sub>Rhl</sub> constructs were negative. Therefore the PCR was repeated later with new colonies.  
+
<img alt="August 2" src="https://static.igem.org/mediawiki/2013/c/cb/Braunschweig_Lab_Journal_August_2.png" width="250"  vspace="20" align="left" style="margin-right:5px"/>In order to test the success of the last days’ cloning experiment we conducted a colony PCR. Unfortunately all clones of both our supposed final Prhl constructs were negative. Therefore the PCR was repeated later with new colonies.  
-
The N-buturyl-HSL synthase RhlI with amilGFP reporter cassette (B0032-C0070-B0015-J23100-B0032-K592010-B0015) and the eCFP cassette (J23100-E0420) showed both clones bearing the correct construct.<br>
+
The N-butyryl-HSL synthase RhlI with amilGFP reporter cassette (B0032-C0070-B0015-J23100-B0032-K592010-B0015) and the eCFP cassette (J23100-E0420) showed both clones bearing the correct construct.<br>
We also conducted a test in 5 mL 2xYT liquid cultures with different concentrations of ampicillin and autoinducers to test if our new constructs were leaky. Cells bearing the inducible ampicillin resistance and LasR transcription factor (B0015-K091117-B0032-ampR-B0015-J23100-B0032-C0079) were tested with ampicillin supplements between 1 and 8 µg/mL. We expected no growth as the ampicillin resistance was not induced.<br>
We also conducted a test in 5 mL 2xYT liquid cultures with different concentrations of ampicillin and autoinducers to test if our new constructs were leaky. Cells bearing the inducible ampicillin resistance and LasR transcription factor (B0015-K091117-B0032-ampR-B0015-J23100-B0032-C0079) were tested with ampicillin supplements between 1 and 8 µg/mL. We expected no growth as the ampicillin resistance was not induced.<br>
Cells carrying the inducible ampicillin resistance with RhlR expression cassette (R0071-B0032-B0015-J23100-B0032-C0071) were incubated in 2xYT medium containing ampicillin in concentrations between 1 and 2 µg/mL and additionally 5 to 50 µmol/L N-butyryl-HSL (Rhl autoinducer). As we induce the ampicillin resistance we expect some of the cultures to grow, depending on which concentration is optimal.<br>
Cells carrying the inducible ampicillin resistance with RhlR expression cassette (R0071-B0032-B0015-J23100-B0032-C0071) were incubated in 2xYT medium containing ampicillin in concentrations between 1 and 2 µg/mL and additionally 5 to 50 µmol/L N-butyryl-HSL (Rhl autoinducer). As we induce the ampicillin resistance we expect some of the cultures to grow, depending on which concentration is optimal.<br>
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     <h2><a href="#Week12">Week 12: August 4 - August 10, 2013</a></h2>
     <h2><a href="#Week12">Week 12: August 4 - August 10, 2013</a></h2>
     <p><p style=" margin-left:5px; margin-right:5px;">
     <p><p style=" margin-left:5px; margin-right:5px;">
-
This week was overshadowed by contaminated medium interfering with our experiments. We managed to transform the final P<sub>Rhl</sub> inducible construct in <i>E. coli</i> Top10F’. As the promoter seemed to be not leaky in this strain we successfully conducted a growth curve experiment showing a difference in growth between induced and non-induced cultures. We are still missing our final P<sub>Las</sub>L inducible construct.</p>
+
This week was overshadowed by contaminated medium interfering with our experiments. We managed to transform the final Prhl inducible construct in <i>E. coli</i> Top10F’. As the promoter seemed to be not leaky in this strain we successfully conducted a growth curve experiment showing a difference in growth between induced and non-induced cultures. We are still missing our final Plas inducible construct.</p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Kevin, Anna, Melanie</b><br>
<b>Investigators: Kevin, Anna, Melanie</b><br>
-
The successful cloning of our final P<sub>Rhl</sub> inducible construct was finally confirmed via colony PCR. Unfortunately the liquid cultures for the minipreps were contaminated. We decided to repeat the entire colony PCR as last PCR’s replated colonies showed almost no coloration.<br>
+
The successful cloning of our final Prhl inducible construct was finally confirmed via colony PCR. Unfortunately the liquid cultures for the minipreps were contaminated. We decided to repeat the entire colony PCR as last PCR’s replated colonies showed almost no coloration.<br>
-
We also adapted a new cloning strategy for our P<sub>Las</sub> inducible construct by swapping vector and insert. As we discovered later, the insert was extremely hard to recover via gel extraction because of almost identical band sizes. We still tried it and proceeded with ligation.<br>
+
We also adapted a new cloning strategy for our Plas inducible construct by swapping vector and insert. As we discovered later, the insert was extremely hard to recover via gel extraction because of almost identical band sizes. We still tried it and proceeded with ligation.<br>
-
We prepared liquid cultures for a miniprep of the final P<sub>Rhl</sub> inducible construct, the N-butyryl-HSL synthase RhlI with amilGFP reporter cassette (B0032-C0070-B0015-J23100-B0032-K592010-B0015) and the eCFP cassette (J23100- E0420). These were, again, contaminated the next day.<br>
+
We prepared liquid cultures for a miniprep of the final Prhl inducible construct, the N-butyryl-HSL synthase RhlI with amilGFP reporter cassette (B0032-C0070-B0015-J23100-B0032-K592010-B0015) and the eCFP cassette (J23100- E0420). These were, again, contaminated the next day.<br>
-
In order to find a setup where our promotors are not leaky, we conducted an experiment with liquid cultures of cells bearing the P<sub>Rhl</sub> inducible ampicillin resistance with RhlR transcription factor cassette (R0071-B0032-B0015-J23100-B0032-C0071) and the P<sub>Las</sub> inducible construct ampicillin resistance combined with LasR transcription factor cassette (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) in which we varied the concentration of ampicillin and the corresponding inducer. The next day these cultures were contaminated like the rest of today's experiments. This was a serious problem!<br>
+
In order to find a setup where our promotors are not leaky, we conducted an experiment with liquid cultures of cells bearing the Prhl inducible ampicillin resistance with RhlR transcription factor cassette (R0071-B0032-B0015-J23100-B0032-C0071) and the Plas inducible construct ampicillin resistance combined with LasR transcription factor cassette (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) in which we varied the concentration of ampicillin and the corresponding inducer. The next day these cultures were contaminated like the rest of today's experiments. This was a serious problem!<br>
In order to test if the leaky promotor is caused by the <i>E. coli</i> XL1 Blue MRF' strain we transformed the inducible ampicillin resistance in <i>E. coli</i> Top10F’.</p>
In order to test if the leaky promotor is caused by the <i>E. coli</i> XL1 Blue MRF' strain we transformed the inducible ampicillin resistance in <i>E. coli</i> Top10F’.</p>
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<b>Investigators: Kevin, Anna</b><br>
<b>Investigators: Kevin, Anna</b><br>
We managed to test our inducible resistances without contamination interfering with the experiment:
We managed to test our inducible resistances without contamination interfering with the experiment:
-
– Cells bearing the the P<sub>Rhl</sub> inducible ampicillin resistance combined with LasR transcription factor and added terminator (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) were tested with ampicillin supplements between 1 and 8 µg/mL. We expected no growth as the ampicillin resistance was not induced.<br>
+
– Cells bearing the the Prhl inducible ampicillin resistance combined with LasR transcription factor and added terminator (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) were tested with ampicillin supplements between 1 and 8 µg/mL. We expected no growth as the ampicillin resistance was not induced.<br>
-
– Cells carrying the P<sub>Rhl</sub> inducible ampicillin resistance combined with LasR transcription factor and added terminator (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) were cultivated on medium containing ampicillin supplements between 1 and 8 µg/mL and additionally 10 µmol/ml N-3-oxododecanoyl-homoserine lactone. As we induced the ampicillin resistance we expected some of the cultures to grow, depending on which concentration is optimal.
+
– Cells carrying the Prhl inducible ampicillin resistance combined with LasR transcription factor and added terminator (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) were cultivated on medium containing ampicillin supplements between 1 and 8 µg/mL and additionally 10 µmol/ml N-3-oxododecanoyl-homoserine lactone. As we induced the ampicillin resistance we expected some of the cultures to grow, depending on which concentration is optimal.
-
– Cells bearing the P<sub>Rhl</sub> inducible ampicillin resistance with RhlR expression cassette (R0071-B0032-B0015-J23100-B0032-C0071) were tested with ampicillin supplements between 1 and 8 µg/mL. We expected no growth as the ampicillin resistance was not induced.<br>
+
– Cells bearing the Prhl inducible ampicillin resistance with RhlR expression cassette (R0071-B0032-B0015-J23100-B0032-C0071) were tested with ampicillin supplements between 1 and 8 µg/mL. We expected no growth as the ampicillin resistance was not induced.<br>
-
– Cells bearing the P<sub>Rhl</sub> inducible ampicillin resistance with RhlR expression cassette (R0071-B0032-B0015-J23100-B0032-C0071) were cultivated on medium containing ampicillin supplements between 1 and 8 µg/mL and additionally 10 µmol/mL N-butyryl-homoserine lactone. As we induce the ampicillin resistance we expect some of the cultures to grow, depending on which concentration is optimal.<br>
+
– Cells bearing the Prhl inducible ampicillin resistance with RhlR expression cassette (R0071-B0032-B0015-J23100-B0032-C0071) were cultivated on medium containing ampicillin supplements between 1 and 8 µg/mL and additionally 10 µmol/mL N-butyryl-homoserine lactone. As we induce the ampicillin resistance we expect some of the cultures to grow, depending on which concentration is optimal.<br>
– Cells carrying the inducible ampicillin resistance combined with LasR transcription factor cassette (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) and the inducible ampicillin resistance with RhlR expression cassette (R0071-B0032-B0015-J23100-B0032-C0071) respectively were grown in media supplemented with chloramphenicol as positive control. Medium supplemented with ampicillin only and medium supplemented with each inducer only were prepared as negative controls.<br>
– Cells carrying the inducible ampicillin resistance combined with LasR transcription factor cassette (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) and the inducible ampicillin resistance with RhlR expression cassette (R0071-B0032-B0015-J23100-B0032-C0071) respectively were grown in media supplemented with chloramphenicol as positive control. Medium supplemented with ampicillin only and medium supplemented with each inducer only were prepared as negative controls.<br>
The next day all but the negative controls showed normal growth confirming that our promotor is still leaky.<br><br>
The next day all but the negative controls showed normal growth confirming that our promotor is still leaky.<br><br>
-
A colony PCR confirmed that transformation of the P<sub>Las</sub> inducible ampicillin resistance with LasR transcription factor cassette (B0015-K091117-B0032-ampR-B0015-J23100-B0032-C0079) and the P<sub>Rhl</sub> inducible construct ampicillin resistance with RhlR expression cassette (R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) into Top10F’ was successful.</p>
+
A colony PCR confirmed that transformation of the Plas inducible ampicillin resistance with LasR transcription factor cassette (B0015-K091117-B0032-ampR-B0015-J23100-B0032-C0079) and the Prhl inducible construct ampicillin resistance with RhlR expression cassette (R0071-B0032-<i>ampR</i>-B0015-J23100-B0032-C0071) into Top10F’ was successful.</p>
<p><img alt="August 6" src="https://static.igem.org/mediawiki/2013/c/ca/Braunschweig_Lab_Journal_August_6.png" width="600"  vspace="20" align="center"/></p>
<p><img alt="August 6" src="https://static.igem.org/mediawiki/2013/c/ca/Braunschweig_Lab_Journal_August_6.png" width="600"  vspace="20" align="center"/></p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">New liquid cultures for prepping the final P<sub>Rhl</sub> inducible construct (K1073035), the N-butyryl-HSL synthase RhlI with amilGFP reporter cassette (B0032-C0070-B0015-J23100-B0032-K592010-B0015) and the eCFP cassette (J23100-E0420) in <i>E. coli</i> TOP10F’ and <i>E. coli</i> XL1 BlueMRF' were inoculated. </p>
+
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">New liquid cultures for prepping the final Prhl inducible construct (K1073035), the N-butyryl-HSL synthase RhlI with amilGFP reporter cassette (B0032-C0070-B0015-J23100-B0032-K592010-B0015) and the eCFP cassette (J23100-E0420) in <i>E. coli</i> TOP10F’ and <i>E. coli</i> XL1 BlueMRF' were inoculated. </p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Jan, Kevin, Laura</b><br>
<b>Investigators: Jan, Kevin, Laura</b><br>
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We retried to clone the final P<sub>Las</sub> inducible construct containing the aeBlue chromoprotein and added a new construct to our list as an alternative to the final P<sub>Rhl</sub> inducible construct containing the eforRed chromoprotein because the aeBlue chromoprotein is not usable for our planned fluorescence experiments.
+
We retried to clone the final Plas inducible construct containing the aeBlue chromoprotein and added a new construct to our list as an alternative to the final Prhl inducible construct containing the eforRed chromoprotein because the aeBlue chromoprotein is not usable for our planned fluorescence experiments.
-
The final P<sub>Las</sub> inducible construct was derived from the N-butyryl-HSL synthetase RhlI with aeBlue reporter cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015) (vector) and the P<sub>Las</sub> inducible ampicillin resistance with LasR transcription factor cassette (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) (insert).
+
The final Plas inducible construct was derived from the N-butyryl-HSL synthetase RhlI with aeBlue reporter cassette (B0032-C0070-B0015-J23100-B0032-K864401-B0015) (vector) and the Plas inducible ampicillin resistance with LasR transcription factor cassette (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) (insert).
-
The final P<sub>Las</sub> inducible construct with amilGFP reporter (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079-B0032-C0070-B0015-J23100-B0032-K592010-B0015) was derived from the N-butyryl-HSL synthetase RhlI with amilGFP reporter cassette (B0032-C0070-B0015-J23100-B0032-K592010-B0015) (vector) and the Las-inducible ampicillin resistance with LasR transcrip-tion factor (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) (insert).<br><br>
+
The final Plas inducible construct with amilGFP reporter (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079-B0032-C0070-B0015-J23100-B0032-K592010-B0015) was derived from the N-butyryl-HSL synthetase RhlI with amilGFP reporter cassette (B0032-C0070-B0015-J23100-B0032-K592010-B0015) (vector) and the Las-inducible ampicillin resistance with LasR transcrip-tion factor (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079) (insert).<br><br>
To test whether our promotor is still leaky in <i>E. coli</i> Top10F' we cultivated our newly transformed strains in 2xYT medium supplemented with 1 µg/mL ampicillin. Normal growth in all cultures showed that the promotor is leaky in this strain as well.<br><br>
To test whether our promotor is still leaky in <i>E. coli</i> Top10F' we cultivated our newly transformed strains in 2xYT medium supplemented with 1 µg/mL ampicillin. Normal growth in all cultures showed that the promotor is leaky in this strain as well.<br><br>
We send our freshly prepped bricks for sequencing. As a result the sequences of the final Prhl the N-butyryl-HSL synthetase RhlI with amilGFP reporter cassette (B0032-C0070-B0015-J23100-B0032-K592010-B0015) was confirmed, but the prepped DNA were contaminated.  <br>  
We send our freshly prepped bricks for sequencing. As a result the sequences of the final Prhl the N-butyryl-HSL synthetase RhlI with amilGFP reporter cassette (B0032-C0070-B0015-J23100-B0032-K592010-B0015) was confirmed, but the prepped DNA were contaminated.  <br>  
-
The sequence of the eCFP cassette (J23100-E0420) was confirmed, but the prepped DNA was also contaminated as well as the DNA of the P<sub>Las</sub> inducible ampicillin resistance with RhlR expression cassette (R0071-B0032-B0015-J23100-B0032-C0071).  </p>
+
The sequence of the eCFP cassette (J23100-E0420) was confirmed, but the prepped DNA was also contaminated as well as the DNA of the Plas inducible ampicillin resistance with RhlR expression cassette (R0071-B0032-B0015-J23100-B0032-C0071).  </p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Kerstin, Laura</b><br>
<b>Investigators: Kerstin, Laura</b><br>
-
<img alt="August 8" src="https://static.igem.org/mediawiki/2013/c/cb/Braunschweig_Lab_Journal_August_8.png" width="400"  vspace="20" align="right"/>A colony PCR showed the expected bands for the final P<sub>Las</sub> inducible construct (K1073034) and the final P<sub>Las</sub> inducible construct with amilGFP reporter (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079-B0032-C0070-B0015-J23100-B0032-K592010-B0015). Hopefully our final constructs are ready to use now. The clones were prepped for sequencing.</p>
+
<img alt="August 8" src="https://static.igem.org/mediawiki/2013/c/cb/Braunschweig_Lab_Journal_August_8.png" width="400"  vspace="20" align="right"/>A colony PCR showed the expected bands for the final Plas inducible construct (K1073034) and the final Plas inducible construct with amilGFP reporter (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079-B0032-C0070-B0015-J23100-B0032-K592010-B0015). Hopefully our final constructs are ready to use now. The clones were prepped for sequencing.</p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Kerstin, Laura</b><br>
<b>Investigators: Kerstin, Laura</b><br>
-
The final P<sub>Rhl</sub> inducible construct (K1073035) was retransformed into <i>E. coli</i> Top10F'.<br>
+
The final Prhl inducible construct (K1073035) was retransformed into <i>E. coli</i> Top10F'.<br>
-
The prepped DNA of the final P<sub>Las</sub> inducible construct (K1073034) and the P<sub>Las</sub> inducible amilGFP cassette (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079-B0032-C0070-B0015-J23100-B0032-K592010-B0015) was sequenced. While the sequence of the P<sub>Las</sub> inducible amilGFP cassette could not be verified, the sequence of the final P<sub>Las</sub> inducible construct containing aeBlue expression cassette was finally verified!<br>
+
The prepped DNA of the final Plas inducible construct (K1073034) and the Plas inducible amilGFP cassette (B0015-K091117-B0032-<i>ampR</i>-B0015-J23100-B0032-C0079-B0032-C0070-B0015-J23100-B0032-K592010-B0015) was sequenced. While the sequence of the Plas inducible amilGFP cassette could not be verified, the sequence of the final Plas inducible construct containing aeBlue expression cassette was finally verified!<br>
-
We prepared liquid cultures of the final P<sub>Rhl</sub> construct (K1073035) in different <i>E. coli</i> strains (XL1 Blue MRF’, Top10F') for a growth curve experiment.</p>
+
We prepared liquid cultures of the final Prhl construct (K1073035) in different <i>E. coli</i> strains (XL1 Blue MRF’, Top10F') for a growth curve experiment.</p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Kerstin, Laura</b><br>
<b>Investigators: Kerstin, Laura</b><br>
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<img alt="August 10" src="https://static.igem.org/mediawiki/parts/thumb/e/e4/Braunschweig2013_Top10_pSB1C3_K1073035.jpg/500px-Braunschweig2013_Top10_pSB1C3_K1073035.jpg" width="350" vspace="20" align="right"/>Some of the liquid culture was used for prepping and sequencing the P<sub>Rhl</sub> inducible construct in <i>E. coli</i> Top10F' cells. The sequence was confirmed.<br>
+
<img alt="August 10" src="https://static.igem.org/mediawiki/parts/thumb/e/e4/Braunschweig2013_Top10_pSB1C3_K1073035.jpg/500px-Braunschweig2013_Top10_pSB1C3_K1073035.jpg" width="350" vspace="20" align="right"/>Some of the liquid culture was used for prepping and sequencing the Prhl inducible construct in <i>E. coli</i> Top10F' cells. The sequence was confirmed.<br>
-
We measured our first successful growth curve showing the difference in growth between induced and non-induced versions of the P<sub>Rhl</sub> inducible construct. The eforRed expression cassette in <i>E. coli</i> TOP10F' was miniprepped and glycerol stocks of this strain were made.
+
We measured our first successful growth curve showing the difference in growth between induced and non-induced versions of the Prhl inducible construct. The eforRed expression cassette in <i>E. coli</i> Top10F' was miniprepped and glycerol stocks of this strain were made.
-
The main cultures (induced and not induced) of the final PRhl inducible construct were inoculated to a start OD520 of 0.05 in 75 ml 2xYT containing ampicillin with cells of the pre-culture. In order to induce the expression of ampR N-3-buturyl homoserine lactone was added to a final concentration of 10 µM. Samples were taken at appropriate times depending on the growth phase until the induced culture reached the stationary phase. OD was determined at 520 nm to avoid absorptions by chromoproteins.
+
The main cultures (induced and not induced) of the final PRhl inducible construct were inoculated to a start OD520 of 0.05 in 75 ml 2xYT containing ampicillin with cells of the pre-culture. In order to induce the expression of ampR N-3-butyryl homoserine lactone was added to a final concentration of 10 µM. Samples were taken at appropriate times depending on the growth phase until the induced culture reached the stationary phase. OD was determined at 520 nm to avoid absorptions by chromoproteins.
<br>
<br>
</div>
</div>
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During the day we noticed that the 2xYT medium used for the pre-cultures yesterday showed contamination. Thus, there is a high possibility  of contamination in the pre-culture as well as in the main culture. Therefore we decided to repeat the experiment. New pre-cultures of the three chromoprotein expression cassettes where inoculated in 50 ml 2xYT containing chloramphenicol directly from -80°C.<br>
During the day we noticed that the 2xYT medium used for the pre-cultures yesterday showed contamination. Thus, there is a high possibility  of contamination in the pre-culture as well as in the main culture. Therefore we decided to repeat the experiment. New pre-cultures of the three chromoprotein expression cassettes where inoculated in 50 ml 2xYT containing chloramphenicol directly from -80°C.<br>
In order to have our constructs available in different <i>E. coli</i> strains for the fluorescence microscopy experiments, the eforRed expression cassette was transformed into <i>E. coli</i> Top10F' by electroporation. Transformed cells were plated on 2xYT agar containing chloramphenicol and incubated over night at 37°C.<br>
In order to have our constructs available in different <i>E. coli</i> strains for the fluorescence microscopy experiments, the eforRed expression cassette was transformed into <i>E. coli</i> Top10F' by electroporation. Transformed cells were plated on 2xYT agar containing chloramphenicol and incubated over night at 37°C.<br>
-
A continuous cultivation of the P<sub>Rhl</sub> inducible in <i>E. coli</i> Top10F' was prepared, including preparation of pre-cultures of our inducible construct in 50 ml 2xYT containing chloramphenicol and grown at 37°C and 250 rpm over night.<br>
+
A continuous cultivation of the Prhl inducible in <i>E. coli</i> Top10F' was prepared, including preparation of pre-cultures of our inducible construct in 50 ml 2xYT containing chloramphenicol and grown at 37°C and 250 rpm over night.<br>
  </p>
  </p>
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The reporter strains for the Las and Rhl systems (<i>E. coli</i> JM109 pSB1075 and <i>E. coli</i> JM109 pSB406 respectively) arrived today. Liquid cultures in 2xYT containing ampicillin were inoculated and grown at 37°C and 250 rpm overnight.<br>  
The reporter strains for the Las and Rhl systems (<i>E. coli</i> JM109 pSB1075 and <i>E. coli</i> JM109 pSB406 respectively) arrived today. Liquid cultures in 2xYT containing ampicillin were inoculated and grown at 37°C and 250 rpm overnight.<br>  
-
Pre-cultures of <i>E. coli</i> Top10F' containing final P<sub>Rhl</sub> inducible construct and <i>E. coli</i> XL1 Blue MRF' containing final P<sub>Las</sub> inducible construct in 50 ml 2xYT containing chloramphenicol for continuous cultures were grown at 37°C and 250 rpm overnight.<br></p>  
+
Pre-cultures of <i>E. coli</i> Top10F' containing final Prhl inducible construct and <i>E. coli</i> XL1 Blue MRF' containing final Plas inducible construct in 50 ml 2xYT containing chloramphenicol for continuous cultures were grown at 37°C and 250 rpm overnight.<br></p>  
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Pre-culture of erforRed expression cassette in <i>E. coli</i> Top10F' in 50 ml 2xYT containing chloramphenicol was inoculated from the  agar plate and grown at 37°C and 250 rpm overnight.<br>
Pre-culture of erforRed expression cassette in <i>E. coli</i> Top10F' in 50 ml 2xYT containing chloramphenicol was inoculated from the  agar plate and grown at 37°C and 250 rpm overnight.<br>
-
For the reproduction of the growth curve a pre-culture of the P<sub>Rhl</sub> inducible construct in <i>E. coli</i> Top10F' in 30 mL 2xYT containing chloramphenicol and incubated at 37°C overnight. <br>
+
For the reproduction of the growth curve a pre-culture of the Prhl inducible construct in <i>E. coli</i> Top10F' in 30 mL 2xYT containing chloramphenicol and incubated at 37°C overnight. <br>
To test the production of homoserine lactones (HSL) by our final constructs we prepared a pre-culture of each reporter strain in 30 ml 2xYT containing ampicillin and incubated them overnight at 37°C and 250 rpm. We also made glycerol cell stocks of the reporter strains for further use.<br>
To test the production of homoserine lactones (HSL) by our final constructs we prepared a pre-culture of each reporter strain in 30 ml 2xYT containing ampicillin and incubated them overnight at 37°C and 250 rpm. We also made glycerol cell stocks of the reporter strains for further use.<br>
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The eforRed expression cassette in <i>E. Coli</i> TOP10F’ was miniprepped and glycerol stocks of this strain were made.<br>
The eforRed expression cassette in <i>E. Coli</i> TOP10F’ was miniprepped and glycerol stocks of this strain were made.<br>
-
The main cultures (induced and not induced) of the final P<sub>Rhl</sub> inducible construct were inoculated to a start OD<sub>520</sub> of 0.05 in 75 ml 2xYT containing ampicillin with cells of the pre-culture. In order to induce the expression of <i>ampR</i> N-3-buturyl homoserine lactone was added to a final concentration of 10 µM. Samples were taken at appropriate times depending on the growth phase until the induced culture reached the stationary phase. OD was determined at 520 nm to avoid absorptions by chromoproteins.<br>
+
The main cultures (induced and not induced) of the final Prhl inducible construct were inoculated to a start OD<sub>520</sub> of 0.05 in 75 ml 2xYT containing ampicillin with cells of the pre-culture. In order to induce the expression of <i>ampR</i> N-3-butyryl homoserine lactone was added to a final concentration of 10 µM. Samples were taken at appropriate times depending on the growth phase until the induced culture reached the stationary phase. OD was determined at 520 nm to avoid absorptions by chromoproteins.<br>
To verify production of HSLs by our constructs the pre-cultures containing the finale constructs as well as the negative controls were centrifuged for 10 min at 6000 rpm and 4°C. Supernatant was transferred to a new Falcon tube and sterilized by filtration. <br>
To verify production of HSLs by our constructs the pre-cultures containing the finale constructs as well as the negative controls were centrifuged for 10 min at 6000 rpm and 4°C. Supernatant was transferred to a new Falcon tube and sterilized by filtration. <br>
Dilution series of the supernatants and the synthetic HSLs as standards were pipetted in 96-well microtiter plates. Wells were inoculated with the corresponding reporter strain and grown for 3 h at 37°C. Bioluminescence produced by the <i>luxCDABE</i> of the reporter strains was detected by a microplate reader. We were able to show that our constructs produced the specific HSLs. However due to the high background especially in the N-3-oxododecanoyl-HSL producing strain we might need to modify the experimental layout in order to get a stronger signal.<br><br><br></p>
Dilution series of the supernatants and the synthetic HSLs as standards were pipetted in 96-well microtiter plates. Wells were inoculated with the corresponding reporter strain and grown for 3 h at 37°C. Bioluminescence produced by the <i>luxCDABE</i> of the reporter strains was detected by a microplate reader. We were able to show that our constructs produced the specific HSLs. However due to the high background especially in the N-3-oxododecanoyl-HSL producing strain we might need to modify the experimental layout in order to get a stronger signal.<br><br><br></p>
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     <h2><a href="#Week14">Week 14: August 18 - August 24, 2013</a></h2>
     <h2><a href="#Week14">Week 14: August 18 - August 24, 2013</a></h2>
     <p><p style=" margin-left:5px; margin-right:5px;">
     <p><p style=" margin-left:5px; margin-right:5px;">
-
<img alt="Conti cultivation" src="https://2013.igem.org/File:Braunschweig_Lab_Week14_continuous_cultivation.jpg" height="200px" align="left" vspace="0" hspace="10" style="margin-right:5px"/>
+
<img alt="Chromoprotein expression" src="https://static.igem.org/mediawiki/2013/7/75/Braunschweig_Lab_Week14_continuous_cultivation.jpg" height="140px" align="left" vspace="0" hspace="10" style="margin-right:5px"/>
The focus of this week’s work was the preparation of the final constructs in order to take pictures under the fluorescence microscope. Furthermore we started the second continuous bioreactor culture.
The focus of this week’s work was the preparation of the final constructs in order to take pictures under the fluorescence microscope. Furthermore we started the second continuous bioreactor culture.
</p>
</p>
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We made glycerol stocks of J23100-B0032-eforRed-cassette and the final inducible constructs in different strains (<i>E. coli</i> Top10 F' and <i>E. coli</i> JM109). Furthermore we transformed J23100-B0032-aeBlue into strain JM109. The functionality of the final Plas construct in strain <i>E. coli</i> JM109 was evaluated by growing it on media supplemented with ampicillin and the inducer N-3-oxododecanoyl homoserine lactone. The strain grew and developed a blue color. This observation led to the conclusion that the construct worked in<i>E. coli</i> JM109.<br>  
We made glycerol stocks of J23100-B0032-eforRed-cassette and the final inducible constructs in different strains (<i>E. coli</i> Top10 F' and <i>E. coli</i> JM109). Furthermore we transformed J23100-B0032-aeBlue into strain JM109. The functionality of the final Plas construct in strain <i>E. coli</i> JM109 was evaluated by growing it on media supplemented with ampicillin and the inducer N-3-oxododecanoyl homoserine lactone. The strain grew and developed a blue color. This observation led to the conclusion that the construct worked in<i>E. coli</i> JM109.<br>  
Furthermore we miniprepped the final Plas contruct in <i>E. coli</i> JM109 and revised the sequencing results of the final eforRed-construct in <i>E. coli</i> Top10 F’. The final Prhl construct was sequence verified.<br>
Furthermore we miniprepped the final Plas contruct in <i>E. coli</i> JM109 and revised the sequencing results of the final eforRed-construct in <i>E. coli</i> Top10 F’. The final Prhl construct was sequence verified.<br>
-
We also conducted regulated and unregulated continuous bioreactor cultivations of mixed cultures of <i>E. coli</i> TOP10F'bearing final Prhl construct and <i>E. coli</i> JM109 containing the final Plas construct. The cultures were inoculated with a 70 % : 30 % ratio of <i>E. coli</i> Top10F' (Prhl construct)/<i>E. coli</i>(Plas construct) and samples were taken every hour. In order to determine the ratios of the strains during the cultivation, the samples were spread out on agar plates and incubated at 37°C. The cultivation was stopped after 25 h. The colonies on the plates were counted as shown below.<p>
+
We also conducted regulated and unregulated continuous bioreactor cultivations of mixed cultures of <i>E. coli</i> TOP10F'bearing final Prhl construct and <i>E. coli</i> JM109 containing the final Plas construct. The cultures were inoculated with a 70 % : 30 % ratio of <i>E. coli</i> Top10F' (Prhl construct)/<i>E. coli</i>(Plas construct) and samples were taken every hour. In order to determine the ratios of the strains during the cultivation, the samples were spread out on agar plates and incubated at 37°C. The cultivation was stopped after 25 h. </p>
-
<p><img alt="August 19" src="https://static.igem.org/mediawiki/2013/a/ae/Braunschweig_Lab_Journal_August_19.png" width="340" vspace="20" align="left"/><img alt="August 19" src="https://static.igem.org/mediawiki/2013/e/e6/Braunschweig_Lab_Journal_August_19_2.png" width="400" vspace="20" align="left"/></p>
+
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We made glycerol stocks of the final Prhl (<i>E. coli</i> Top10F'::K1073035) and Plas (<i>E. coli</i> JM109::K1073034) constructs from agar plates of the reactor cultivation.<br>  
We made glycerol stocks of the final Prhl (<i>E. coli</i> Top10F'::K1073035) and Plas (<i>E. coli</i> JM109::K1073034) constructs from agar plates of the reactor cultivation.<br>  
Furthermore 10 mM stock solutions of the homoserine lactones (autoinducers) of the rhl and las quorum sensing system (1.7 mg N-butyryl  homoserine lactone + 1 ml DMSO and 2.8 mg N-3-oxododecanoyl  homoserine lactone +  933 µl DMSO, respectively) were prepared.<br>
Furthermore 10 mM stock solutions of the homoserine lactones (autoinducers) of the rhl and las quorum sensing system (1.7 mg N-butyryl  homoserine lactone + 1 ml DMSO and 2.8 mg N-3-oxododecanoyl  homoserine lactone +  933 µl DMSO, respectively) were prepared.<br>
-
Additionally we repeated the continuous bioreactor cultivation we conducted Monday. Counting the colonies resulted in the diagrams shown below.<br>
+
Additionally we repeated the continuous bioreactor cultivation we conducted Monday.</p>
-
<img alt="August 22" src="https://static.igem.org/mediawiki/2013/8/86/Braunschweig_Lab_Journal_August_22_2.png" width="400" vspace="20" align="left"/><img alt="August 22" src="https://static.igem.org/mediawiki/2013/1/17/Braunschweig_Lab_Journal_August_22_3.png" width="400" vspace="20" align="left"/>
+
-
</p>
+
   </div>
   </div>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Jan, Anna, Melanie</b><br>
<b>Investigators: Jan, Anna, Melanie</b><br>
-
We prepared pre-cultures of <i>E. coli</i> JM109 carrying the final P<sub>Las</sub> construct for the growth curve experiments tomorrow. 2xYT medium containing chloramphenicol was inoculated with cells directely from glycerol stock and incubated at 37°C and 250 rpm over night.</p>
+
We prepared pre-cultures of <i>E. coli</i> JM109 carrying the final Plas construct for the growth curve experiments tomorrow. 2xYT medium containing chloramphenicol was inoculated with cells directely from glycerol stock and incubated at 37°C and 250 rpm over night.</p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Jan, Anna, Melanie</b><br>
<b>Investigators: Jan, Anna, Melanie</b><br>
-
We conducted a cultivation experiment with the <i>E. coli</i> JM109 bearing the final P<sub>Las</sub> construct to test the growth in presence and Absence of synthetic N-3-oxododecanoyl-HSL autoinducer added to the medium. Cultivation was carried out in four 500 mL non-baffled flasks with 75 mL 2xYT medium with ampicillin. In two flasks N-3-oxododecanoyl-HSL was added. Ideally  the flask without N-3-oxododecanoyl-HSL would not show any growth in the beginning until the antibiotic has been depleted. The N-3-oxododecanoyl-HSL in the other flask should induce the quorum sensing controlled ampicillin resistance. Unfortunately all cultures were growing equally fast from the start so the experiment was aborted.</p>
+
We conducted a cultivation experiment with the <i>E. coli</i> JM109 bearing the final Plas construct to test the growth in presence and absence of synthetic N-3-oxododecanoyl-HSL autoinducer added to the medium. Cultivation was carried out in four 500 mL non-baffled flasks with 75 mL 2xYT medium with Ampicillin. In two flasks N-3-oxododecanoyl-HSL was added. Ideally  the flask without N-3-oxododecanoyl-HSL would not show any growth in the beginning until the antibiotic has been depleted. The N-3-oxododecanoyl-HSL in the other flask should induce the quorum sensing controlled ampicillin resistance. Unfortunately all cultures were growing equally fast from the start so the experiment was aborted.</p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Jan, Anna, Melanie</b><br>
<b>Investigators: Jan, Anna, Melanie</b><br>
-
We repeated the growth curve experiment with our <i>E. coli</i> JM109 bearing final P<sub>Las</sub> to see if the unexpected growth was caused by a resistant contamination. Cultivation was again carried out in four 500 mL non-baffled flasks with 75 mL 2xYT medium with ampicillin. Again, in two out of the four flasks N-3-oxododecanoyl-HSL was added.  The result was the same as yesterday, all culture showed normal growth. We needed a new strategy for future experiments.</p>
+
We repeated the growth curve experiment with our <i>E. coli</i> JM109 bearing final Plas to see if the unexpected growth was caused by a resistant contamination. Cultivation was again carried out in four 500 mL non-baffled flasks with 75 mL 2xYT medium with ampicillin. Again, in two out of the four flasks N-3-oxododecanoyl-HSL was added.  The result was the same as yesterday, all culture showed normal growth. We needed a new strategy for future experiments.</p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
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<b>Investigators: Jan, Anna, Melanie</b><br>
<b>Investigators: Jan, Anna, Melanie</b><br>
We figured that maybe our ampicillin resistance was too potent because even the basal expression allowed for normal growth in medium with antibiotic.<br>
We figured that maybe our ampicillin resistance was too potent because even the basal expression allowed for normal growth in medium with antibiotic.<br>
-
We prepared 5 mL liquid cultures of <i>E. coli</i> JM109 bearing final P<sub>Las</sub> and added varying amounts of a beta-lactamase inhibitor (clavulanic acid) to suppress the background expression. Tubes with 1 µg/µL to 100 µg/µL clavulanic acid showed no growth while tubes with 0.001 µg/µL to 0.1 µg/µL clavulanic acid showed normal growth.</p>  
+
We prepared 5 mL liquid cultures of <i>E. coli</i> JM109 bearing final Plas and added varying amounts of a beta-lactamase inhibitor (clavulanic acid) to suppress the background expression. Tubes with 1 µg/µL to 100 µg/µL clavulanic acid showed no growth while tubes with 0.001 µg/µL to 0.1 µg/µL clavulanic acid showed normal growth.</p>  
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     <h2><a href="#Week18">Week 18: September 15 - September 21, 2013</a></h2>
     <h2><a href="#Week18">Week 18: September 15 - September 21, 2013</a></h2>
     <p><p style=" margin-left:5px; margin-right:5px;">
     <p><p style=" margin-left:5px; margin-right:5px;">
-
This week we were happy to find a workaround for a big troublemaker: the background expression of β-lactamase in non-induced state. We determined the critical concentration of β-lactamase inhibitor clavulanic acid to be around 1 µg/ml. Furthermore, we directly applied it to experiments which previously were problematic because of the resistance to ampicillin under non-induced conditions.<br>
+
This week we were happy to find a workaround for a big troublemaker: the background expression of beta-lactamase in non-induced state. We determined the critical concentration of beta-lactamase inhibitor clavulanic acid to be around 1 µg/ml. Furthermore, we directly applied it to experiments which previously were problematic because of the resistance to ampicillin under non-induced conditions.<br>
-
Furthermore, we added our constructs to the iGEM Registry. Therefore our final contructs are from now on referred to as <a href="http://parts.igem.org/Part:BBa_K1073034">K1073034</a> and <a href="http://parts.igem.org/Part:BBa_K1073035">K1073035</a> for final P<sub>Las</sub> construct and final P<sub>Rhl</sub> construct respectively.
+
Furthermore, we added our constructs to the iGEM Registry. Therefore our final contructs are from now on referred to as <a href="http://parts.igem.org/Part:BBa_K1073034">K1073034</a> and <a href="http://parts.igem.org/Part:BBa_K1073035">K1073035</a> for final Plas construct and final Prhl construct respectively.
</p>
</p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Jan</b><br>
<b>Investigators: Jan</b><br>
-
<img alt="September 16" src="https://static.igem.org/mediawiki/2013/2/2f/Braunschweig_Lab_Journal_September_19.png" width="400" vspace="20" align="right"/>This week started with a quite important experiment: Since we experienced background expression of beta-lactamase, our transformed bacteria were resistant on a low level to ampicillin, although cultivated under non-inducing conditions.  We prepared 5mL liquid cultures of JM109::K1073034 and added varying amounts of a beta-lactamase inhibitor (clavulanic acid) to suppress the background expression. Tubes with 1 µg/µL to 100 µg/µL clavulanic acid showed no growth while tubes with 0.001 µg/µL to 0.1 µg/µL clavulanic acid showed normal growth.</p>
+
<img alt="September 16" src="https://static.igem.org/mediawiki/2013/2/2f/Braunschweig_Lab_Journal_September_19.png" width="400" vspace="20" align="right"/>This week started with a quite important experiment: Since we experienced background expression of beta-lactamase, our transformed bacteria were resistant on a low level to ampicillin, although cultivated under non-inducing conditions.  We prepared 5 mL liquid cultures of <i>E. coli</i> JM109::K1073034 and added varying amounts of a beta-lactamase inhibitor (clavulanic acid) to suppress the background expression. Tubes with 1 µg/µL to 100 µg/µL clavulanic acid showed no growth while tubes with 0.001 µg/µL to 0.1 µg/µL clavulanic acid showed normal growth.</p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
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<b>Investigators: Jan</b><br>
<b>Investigators: Jan</b><br>
On Wednesday, we repeated the experiment we did on Monday with a different scale of concentration steps to more precisely determine the best concentration of clavulanic acid for future experiments. Unfortunately, all samples showed growth so no further distinction could be made.<br>
On Wednesday, we repeated the experiment we did on Monday with a different scale of concentration steps to more precisely determine the best concentration of clavulanic acid for future experiments. Unfortunately, all samples showed growth so no further distinction could be made.<br>
-
Hence, we went ahead with the 1 µg/ml of clavulanic acid we determined on Monday to remove background expression of β-lactamase. We applied this information to a certain growth behavior of JM109 bacteria transformed with our final construct (K1073034). Therefore, we cultivated this strain in 2x YT media with ampicillin and clavulanic acid. Out of four cultures, only two contained the inducer necessary for successful induction of ampicillin resistance.</p>
+
Hence, we went ahead with the 1 µg/ml of clavulanic acid we determined on Monday to remove background expression of beta-lactamase. We applied this information to a certain growth behavior of <i>E. coli</i> JM109 bacteria transformed with our final construct (K1073034). Therefore, we cultivated this strain in 2x YT media with ampicillin and clavulanic acid. Out of four cultures, only two contained the inducer necessary for successful induction of ampicillin resistance.</p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Jan</b><br>
<b>Investigators: Jan</b><br>
-
<img alt="September 19" src="https://static.igem.org/mediawiki/parts/thumb/9/98/Braunschweig_2013_-_JM109_pSB1C3_K1073034.jpg/480px-Braunschweig_2013_-_JM109_pSB1C3_K1073034.jpg" width="400" vspace="20" align="right"/>Liquid cultures of 2x YT media with ampicillin and 1 µg/ml clavulanic acid were inoculated at different ratios (based on OD<sub>520</sub>) of JM109::K1073034 and Top10F'::K1073035. Each culture was additionally performed with chloramphenicol instead of ampicillin as unregulated control. Unfortunately the concentration of clavulanic acid was too high and no growth was observed.</p>
+
<img alt="September 19" src="https://static.igem.org/mediawiki/parts/thumb/9/98/Braunschweig_2013_-_JM109_pSB1C3_K1073034.jpg/480px-Braunschweig_2013_-_JM109_pSB1C3_K1073034.jpg" width="400" vspace="20" align="right"/>Liquid cultures of 2x YT media with ampicillin and 1 µg/ml clavulanic acid were inoculated at different ratios (based on OD<sub>520</sub>) of <i>E. coli</i> JM109::K1073034 and <i>E. coli</i> Top10F'::K1073035. Each culture was additionally performed with chloramphenicol instead of ampicillin as unregulated control. Unfortunately the concentration of clavulanic acid was too high and no growth was observed.</p>
  </div>
  </div>
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<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<p style="margin-left:5px; margin-right:5px; margin-bottom:0px; margin-top:0px">
<b>Investigators: Jan</b><br>
<b>Investigators: Jan</b><br>
-
Overnight cultures were inoculated for the continuous cultivation on Monday. Furthermore, we set up the reactors and made sure we are ready to start early next day.</p>
+
Overnight cultures of <i>E. coli</i> JM109::K1073035 and <i>E. coli</i> Top10F'::K1073035 were inoculated in 2xYT for the continuous cultivation on Monday. Furthermore, we set up the reactors and made sure we are ready to start early next day.</p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
<p><img alt="grey line" src="https://static.igem.org/mediawiki/2013/4/4c/Braunschweig_grey_line.png" width="850" height="1" vspace="20"/></p>
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<h1>Our sponsors</h1></p>
<h1>Our sponsors</h1></p>
<img alt="linie rot 8pix hoch" src="https://static.igem.org/mediawiki/2013/0/07/Team_Braunschweig_Red_line.jpg" width="890" height="1" />
<img alt="linie rot 8pix hoch" src="https://static.igem.org/mediawiki/2013/0/07/Team_Braunschweig_Red_line.jpg" width="890" height="1" />
-
<img src="https://static.igem.org/mediawiki/2013/7/73/SponsorenLogosBS210712.png" width="875px" />
+
<img src="https://static.igem.org/mediawiki/2013/9/9e/SponsorenBS.png" width="875px" />
</div>
</div>
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Latest revision as of 13:15, 27 October 2013

Labjournal

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Braunschweig Labbook This is the documentation of our lab work. Achievements of each week are summerized followed by a daily discription of our experiments.

An overview on how we approached this project can be found under Approach. For detailed protocols of certain procedures please refer to Protocols. Attributions are given for each day, however please check our
Attributions section for efforts beyond the lab work.


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Our sponsors

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