Team:Braunschweig/Notebook

From 2013.igem.org

(Difference between revisions)
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<b>Investigators: Kevin, Kerstin, Laura</b><br>
<b>Investigators: Kevin, Kerstin, Laura</b><br>
Today we transformed our freshly prepared competent cells for test purposes. We used the plasmids pUC 18 and pUC19 to calculate the transformation efficiency. 100 pg of DNA were used for each transformation with 50 µl cells.  
Today we transformed our freshly prepared competent cells for test purposes. We used the plasmids pUC 18 and pUC19 to calculate the transformation efficiency. 100 pg of DNA were used for each transformation with 50 µl cells.  
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Additionally, competent cells were plated on ampicillin, kanamycin and chloramphenicol to ensure that the strain is not carrying any corresponding resistances. The plates were incubated at 37 °C overnight.</p>
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Additionally, competent cells were plated on Ampicillin, Kanamycin and Chloramphenicol to ensure that the strain is not carrying any corresponding resistances. The plates were incubated at 37 °C overnight.</p>
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<b>Investigators: Tabea, Oliver, Laura, Kevin</b><br>
<b>Investigators: Tabea, Oliver, Laura, Kevin</b><br>
<img alt="June7" src="https://static.igem.org/mediawiki/2013/7/77/Braunschweig_Lab_Journal_June_7.png" width="250" align="right" vspace="0" hspace="20"/>
<img alt="June7" src="https://static.igem.org/mediawiki/2013/7/77/Braunschweig_Lab_Journal_June_7.png" width="250" align="right" vspace="0" hspace="20"/>
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We transformed our ligations from yesterday using our competent glycerol stocks without prior heat inactivation of T4-ligase. Transformed cells were plated on agar plates containing glucose and Chloramphenicol and were grown at 37 °C over night.<br>
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We transformed our ligations from yesterday using our competent glycerol stocks without prior heat inactivation of T4-ligase. Transformed cells were plated on agar plates containing glucose and Chloramphenicol and were grown at 37 °C overnight.<br>
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To test the success of our ligation beforehand we conducted a colony-PCR (see protocoll colony-PCR, Extension time 30 s) using 1 µL of our untransformed ligation mix of C0061+B0015 and B0032+J23100 as template. The conducted gel electrophoresis was visualized and showed a band of the expected size for B0032+J23100. The band for C0061+B0015 was too small. Further investigation revealed the chosen extension was too short.<br>
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To test the success of our ligation beforehand we conducted a colony PCR(extension time 30 s) using 1 µL of our untransformed ligation mix of C0061+B0015 and B0032+J23100 as template. The conducted gel electrophoresis was visualized and showed a band of the expected size for B0032+J23100. The band for C0061+B0015 was too small. Further investigation revealed the chosen extension was too short.<br>
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We also send some of the Bricks miniprepped on June 3, 2013 for sequencing (primers VR and VF2).</p>
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We also send some of the BioBricks miniprepped on June 3, 2013 for sequencing (primers VR and VF2).</p>
   </div>
   </div>
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<div id="Week4" class="menuSection">
<div id="Week4" class="menuSection">
     <h2><a href="#Week4">Week 4: June 9 - June 15, 2013</a></h2>
     <h2><a href="#Week4">Week 4: June 9 - June 15, 2013</a></h2>
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<p style=" margin-left:5px; margin-right:5px;">This week we confirmed the successful cloning of the constitutive promoter + RBS. Furthermore we confirmed the addition of the TT to the autoinducer synthases and the lactonase. More biobrick sequences were verified. However, we observed some point mutations in the prefix/suffix sequences of some bricks as well as major annotation differences in the biobrick C0061.
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<p style=" margin-left:5px; margin-right:5px;">This week we confirmed the successful cloning of the constitutive promoter + RBS. Furthermore we confirmed the addition of the double terminator to the autoinducer synthases and the lactonase. More BioBrick sequences were verified. However, we observed some point mutations in the prefix/suffix sequences of some bricks as well as major annotation differences in the BioBrick C0061.
</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: Laura, Kerstin </b><br>
<b>Investigators: Laura, Kerstin </b><br>
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We ran a colony PCR of 2 clones of each ligation (transformation from June 6, 2013) and the biobricks P1002 and K091117 (primers VF2 and VR).<br>
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We ran a colony PCR of two clones of each ligation (transformation from June 6, 2013) and the biobricks P1002 and K091117 (primers VF2 and VR).<br>
We also inoculated overnight cultures for plasmid preparations the next day.</p>
We also inoculated overnight cultures for plasmid preparations the next day.</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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We analyzed yesterday’s colony PCR gelelectrophoresis for the expected fragment sizes. All clones were positive(clone 1 of J23100+B0032 and J23106+B0032 did not contain the promoters as later shown by DNA sequencing.
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We analyzed yesterday’s colony PCR gel electrophoresis for the expected fragment sizes. All clones were positive, although clone 1 of J23100+B0032 and J23106+B0032 did not contain the promoters as later shown by DNA sequencing.
<img alt="June10" src="https://static.igem.org/mediawiki/2013/5/50/Braunschweig_Lab_Journal_June_10.png" width="600" align="center" vspace="0" hspace="10"/><br><br>
<img alt="June10" src="https://static.igem.org/mediawiki/2013/5/50/Braunschweig_Lab_Journal_June_10.png" width="600" align="center" vspace="0" hspace="10"/><br><br>
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We isolated the plasmid DNA from all clones with a Miniprep Kit following the manufacturer’s instructions.</p>
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We isolated the plasmid DNA from all clones with a miniprep kit following the manufacturer’s instructions.</p>
<p style="font-size:14px; font-weight:bold; text-decoration:none; border:none; color:#be1e3c; margin-left:5px; margin-right:5px; margin-top:0px; margin-bottom:0px">Tuesday, June 11, 2013</p>
<p style="font-size:14px; font-weight:bold; text-decoration:none; border:none; color:#be1e3c; margin-left:5px; margin-right:5px; margin-top:0px; margin-bottom:0px">Tuesday, June 11, 2013</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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We transformed the chemically competent E.coli XL1 Blue MRF’ with the ligation C0061+B0015 (prepared on June 6, 2013) and the biobrick E0420 (eCFP).<br>
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We transformed the chemically competent <i>E.coli</i> XL1 Blue MRF’ with the ligation C0061+B0015 (prepared on June 6, 2013) and the biobrick E0420 (eCFP).<br>
The plasmid DNA isolated the day before was sent to GATC for sequencing.<br>
The plasmid DNA isolated the day before was sent to GATC for sequencing.<br>
The DNA sequences from June 7, 2013 were analyzed by sequence alignment with the vector maps.
The DNA sequences from June 7, 2013 were analyzed by sequence alignment with the vector maps.
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<b>Investigators: Tabea, Oliver</b><br>
<b>Investigators: Tabea, Oliver</b><br>
<img alt="June10" src="https://static.igem.org/mediawiki/2013/5/52/Braunschweig_Lab_Journal_June_12.png" width="100" align="right" vspace="0" hspace="10"/>
<img alt="June10" src="https://static.igem.org/mediawiki/2013/5/52/Braunschweig_Lab_Journal_June_12.png" width="100" align="right" vspace="0" hspace="10"/>
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The biobrick B0015 was amplified by PCR using the resuspended DNA from the distribution kit as a template. The PCR was successful (expected fragment size was 443 bp).<br>
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The biobrick B0015 was amplified by PCR using the resuspended DNA from the distribution kit as template. The PCR was successful (expected fragment size was 443 bp).<br>
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A colony PCR of 2 clones of each of yesterday’s transformations (E0420 and C0061+B0015) was run (primers VF2 and VR). Overnight cultures of the analyzed clones were inoculated.<br>
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A colony PCR of two clones of each of yesterday’s transformations (E0420 and C0061+B0015) was run (primers VF2 and VR). Overnight cultures of the analyzed clones were inoculated.<br>
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The remaining DNA sequences (from June 7,2013) were analyzed by sequence alignment. While the Biobricks J06702, B0012, C0071, R0062 and R0071 were sequence verified, the biobrick C0062 did not match the sequence, the DNA sequencing for clone 2 failed. Furthermore the sequence alignment of E0240 revealed additional 25 bp after the EcoRI restriction site.The DNA sequence of E0430 was confirmed except for a point mutation (T to A between Not/I and XbaI restriction site) as well as the biobrick J23112 which contained a point mutation in the prefix sequence (T to A).
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The remaining DNA sequences (from June 7,2013) were analyzed by sequence alignment. While the BioBricks J06702, B0012, C0071, R0062 and R0071 were sequence verified, the BioBrick C0062 did not match the sequence, the DNA sequencing for clone 2 failed. Furthermore the sequence alignment of E0240 revealed additional 25 bp after the EcoRI restriction site. The DNA sequence of E0430 was confirmed except for a point mutation (T to A between Not/I and XbaI restriction site) as well as the BioBrick J23112 which contained a point mutation in the prefix sequence (T to A).
   
   
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<img alt="June13" src="https://static.igem.org/mediawiki/2013/4/41/Braunschweig_Lab_Journal_June_13.png" width="200" align="right" vspace="0" hspace="5"/>
<img alt="June13" src="https://static.igem.org/mediawiki/2013/4/41/Braunschweig_Lab_Journal_June_13.png" width="200" align="right" vspace="0" hspace="5"/>
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>
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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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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 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>
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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>
<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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For C0061, the restriction failed. The size of the linearized vector is about 10kb confirming differences in the partsregistry annotation and the brick.<br>
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For C0061, the restriction failed. The size of the linearized vector is about 10kb confirming differences in the Registry of Standard Parts annotation and the BioBrick.<br>
The sequence data from June 11, 2013 was analyzed by sequence alignments. J23100+B0032 and J23105+B0032 did not contain the promoters. J23112+B0032, C0078+B0015, C0070+B0015, C0060+B0015 was sequence verified. Unfortunately the sequencing failed for K091117 and P1002.</p>
The sequence data from June 11, 2013 was analyzed by sequence alignments. J23100+B0032 and J23105+B0032 did not contain the promoters. J23112+B0032, C0078+B0015, C0070+B0015, C0060+B0015 was sequence verified. Unfortunately the sequencing failed for K091117 and P1002.</p>
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   <div id="Week5" class="menuSection">
   <div id="Week5" class="menuSection">
     <h2><a href="#Week5">Week 5: June 16 - June 22, 2013</a></h2>
     <h2><a href="#Week5">Week 5: June 16 - June 22, 2013</a></h2>
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     <p style=" margin-left:5px; margin-right:5px;">This week was dominated by a lot of cloning. We equipped our constitutive and inducible promoters with ribosome binding sites, terminators, an ampicillin resistance gene and a lactonase (not all of them got each of these elements). Additionally, we separated the ampicillin resistance gene from its native promoter in order to make its expression inducible. Although cloning was mostly successful, we were struggling to amplify and purify the <i>luxR</i> brick (C0062), which cost us a lot of time and effort. Unfortunately, it also wasn’t rewarded with success in the end.</p>
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     <p style=" margin-left:5px; margin-right:5px;">This week was dominated by a lot of cloning. We equipped our constitutive and inducible promoters with ribosome binding sites, terminators, an Ampicillin resistance gene and a lactonase (not all of them got each of these elements). Additionally, we separated the Ampicillin resistance gene from its native promoter in order to make its expression inducible. Although cloning was mostly successful, we were struggling to amplify and purify the <i>luxR</i> brick (C0062), which cost us a lot of time and effort. Unfortunately, it also wasn’t rewarded with success in the end.</p>
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<p style=" margin-left:5px; margin-right:5px;">Another important step was to adapt our cloning strategy towards new chromoproteins which we received from iGEM team Uppsala. These allow us to evaluate our cultuvation experiments solely with own equipment, rather than having to find a cell sorter with suitable excitement lasers for all fluorescent proteins.</p>
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<p style=" margin-left:5px; margin-right:5px;">Another important step was to adapt our cloning strategy towards new chromoproteins which we received from iGEM team Uppsala. These allow us to evaluate our cultivation experiments solely with own equipment, rather than having to find a cell sorter with suitable excitement lasers for all fluorescent proteins.</p>
<p style="font-size:14px; font-weight:bold; text-decoration:none; border:none; color:#be1e3c; margin-left:5px; margin-right:5px; margin-top:0px; margin-bottom:0px">Monday, June 17, 2013</p>
<p style="font-size:14px; font-weight:bold; text-decoration:none; border:none; color:#be1e3c; margin-left:5px; margin-right:5px; margin-top:0px; margin-bottom:0px">Monday, June 17, 2013</p>
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<b>Investigators: Kevin, Jan</b><br>
<b>Investigators: Kevin, Jan</b><br>
We performed a colony-PCR to test if cloning from the previous day was successful. Positively tested clones were used to inoculate suspension cultures for DNA preparation. Overnight suspension cultures of B0015- and B0032-transformed cells were used for DNA preparation.<br>
We performed a colony-PCR to test if cloning from the previous day was successful. Positively tested clones were used to inoculate suspension cultures for DNA preparation. Overnight suspension cultures of B0015- and B0032-transformed cells were used for DNA preparation.<br>
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In order to separate the ampicillin resistance gene from its native promoter we performed a Phusion-PCR with appropriate primers on the P1002 brick.</p>
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In order to separate the Ampicillin resistance gene from its native promoter we performed a Phusion-PCR with appropriate primers on the P1002 brick.</p>
<p style="font-size:14px; font-weight:bold; text-decoration:none; border:none; color:#be1e3c; margin-left:5px; margin-right:5px; margin-top:0px; margin-bottom:0px">Wednesday, June 19, 2013</p>
<p style="font-size:14px; font-weight:bold; text-decoration:none; border:none; color:#be1e3c; margin-left:5px; margin-right:5px; margin-top:0px; margin-bottom:0px">Wednesday, June 19, 2013</p>
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<b>Investigators: Laura, Oliver</b><br>
<b>Investigators: Laura, Oliver</b><br>
First, we performed another Phusion-PCR on ampR (P1002 brick) since the first PCR did not yield enough DNA. We also tried to amplify the luxR gene (C0062) through Phusion-PCR from pSB1A2 plasmid. However, not enough DNA was yielded and digestion of the purified PCR product did not show expected bands.<br>
First, we performed another Phusion-PCR on ampR (P1002 brick) since the first PCR did not yield enough DNA. We also tried to amplify the luxR gene (C0062) through Phusion-PCR from pSB1A2 plasmid. However, not enough DNA was yielded and digestion of the purified PCR product did not show expected bands.<br>
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More digestions were set up to harvest the separated ampicillin resistance gene and prepare DNA for the next cloning round. Additionally, lactonase (C0060) was ligated with a RBS (B0032), ampR gene was cloned behind our inducible promoters and we ligated each autoinducer synthetase with a RBS (B0032) over night.
+
More digestions were set up to harvest the separated Ampicillin resistance gene and prepare DNA for the next cloning round. Additionally, lactonase (C0060) was ligated with a RBS (B0032), ampR gene was cloned behind our inducible promoters and we ligated each autoinducer synthetase with a RBS (B0032) over night.
</p>
</p>
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Even if we switched to our new strategy we still keep working on the other fluorescent markers. Therefore we digested the bricks 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. Gelextraction 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>
Even if we switched to our new strategy we still keep working on the other fluorescent markers. Therefore we digested the bricks 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. Gelextraction 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>
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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 leakyness 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 leakyness 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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Before we started working on our own chromoprotein-constructs, we screened for the optimum cultivation conditions. We cultivated E. coli XL1 containing a new construct from Uppsala iGEM containing the device J23110-B0034-aeBlue. We tested expression of the blue chromoprotein at different temperatures, oxygen supply and rpm. From that experiments we came to the conclusion that low oxygen supply and a temperature of 37°C leads to higher expression rates.<br><br>
Before we started working on our own chromoprotein-constructs, we screened for the optimum cultivation conditions. We cultivated E. coli XL1 containing a new construct from Uppsala iGEM containing the device J23110-B0034-aeBlue. We tested expression of the blue chromoprotein at different temperatures, oxygen supply and rpm. From that experiments we came to the conclusion that low oxygen supply and a temperature of 37°C leads to higher expression rates.<br><br>
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The evaluation of the experiment for the inducible promotors showed that only the Prhl is not leaky. Plux as well as Plas were leaky and showed growth of E. coli XL1 at all tested ampicillin concentrations. Therefore we repeated the experiment using higher ampicillin conentrations.<br><br>
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The evaluation of the experiment for the inducible promotors showed that only the Prhl is not leaky. Plux as well as Plas were leaky and showed growth of E. coli XL1 at all tested Ampicillin concentrations. Therefore we repeated the experiment using higher Ampicillin conentrations.<br><br>
The bricks containing the fluorescent markers ligated yesterday were transformed in <i>E. coli</i> XL1 by heatshock.<br><br>
The bricks containing the fluorescent markers ligated yesterday were transformed in <i>E. coli</i> XL1 by heatshock.<br><br>

Revision as of 09:19, 2 October 2013

Labjournal

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This is the documentation of our lab work. 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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