Team:Braunschweig/Notebook

From 2013.igem.org

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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.
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.
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<img alt="August 19" src="https://static.igem.org/mediawiki/2013/a/ae/Braunschweig_Lab_Journal_August_19.png" width="350" 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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<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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<b>Investigators: Kevin</b><br>
<b>Investigators: Kevin</b><br>
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<img alt="August 27" src="https://static.igem.org/mediawiki/2013/a/aa/Braunschweig_Lab_Journal_August_27.jpg" width="400" align="right" vspace="0" hspace="20"/> Today we checked one red, one blue and six white colonies from a spread out sample that we took during our previous continuous cultivation for successful integration of our cloned vectors. This was primarily done to reveal the unknown source of white cultures on our agar plates. The blue colony and three of the white colonies were positive for integration of the aeBlue construct (K1073034). These colonies later turned blue. For some reason, all colonies showed integration of eforRed, including the blue control.<br>
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<img alt="August 27" src="https://static.igem.org/mediawiki/2013/a/aa/Braunschweig_Lab_Journal_August_27.jpg" width="400" align="right" vspace="0" hspace="20"/> Today we checked one pink, one blue and six white colonies from a spread out sample that we took during our previous continuous cultivation for successful integration of our cloned vectors. This was primarily done to reveal the unknown source of white cultures on our agar plates. The blue colony and three of the white colonies were positive for integration of the final Plas construct. These colonies later turned blue. For some reason, all colonies showed integration of final Prhl construct, including the blue control.<br>
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Furthermore, Kevin inoculated liquid cultures of K1073034 and K1073035 constructs in JM109 and Top10 F’ cells and also the autoinducer detecting strains <i>E. coli</i> JM109 pSB1075 and <i>E. coli</i> JM109 pSB406 which are used to detect the production ofautoinducers by our constructs.</p>
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Furthermore, we inoculated liquid cultures of final Plas and final Prhl constructs in <i>E. coli</i> JM109 and <i>E. coli</i> Top10 F' cells and also the autoinducer detecting strains <i>E. coli</i> JM109 pSB1075 and <i>E. coli</i> JM109 pSB406 which are used to detect the production ofautoinducers by our constructs.</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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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 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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Furthermore, we added our constructs to the iGEM Registry. Therefore our final contructs are from now on referred to as K1073034 and K1073035 for final P<sub>Las</sub> construct and final P<sub>Rhl</sub> construct respectively.
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Furthermore, we added our constructs to the iGEM Registry. Therefore our final contructs are from now on referred to as <partinfo>BBa_K1073034</partinfo> and <partinfo>BBa_K1073035</partinfo> for final P<sub>Las</sub> construct and final P<sub>Rhl</sub> construct respectively.
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Revision as of 23:47, 4 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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