Team:Braunschweig/Notebook

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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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     <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.
+
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>
+
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>
-
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>
-
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>
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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>
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<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>  
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<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>  
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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>
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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 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>
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>
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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>  
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>
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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>
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<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 Prhl constructs were negative. Therefore the PCR was repeated later with new colonies.  
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<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.  
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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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<b>Investigators: Kerstin, Laura</b><br>
<b>Investigators: Kerstin, Laura</b><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>
<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>
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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.
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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.
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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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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>
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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-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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<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>
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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>
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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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     <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;">
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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>
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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 Plas construct and final Prhl 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>
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<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>
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<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>
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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>
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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" />
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<img src="https://static.igem.org/mediawiki/2013/7/73/SponsorenLogosBS210712.png" width="875px" />
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<img src="https://static.igem.org/mediawiki/2013/9/9e/SponsorenBS.png" width="875px" />
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Latest revision as of 13:15, 27 October 2013

Labjournal

linie rot 8pix hoch

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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