https://wiki.fkkt.uni-lj.si/index.php?action=history&feed=atom&title=A_synthetic_oscillatory_network_of_transcriptional_regulatorsA synthetic oscillatory network of transcriptional regulators - Revision history2026-04-21T15:37:44ZRevision history for this page on the wikiMediaWiki 1.39.3https://wiki.fkkt.uni-lj.si/index.php?title=A_synthetic_oscillatory_network_of_transcriptional_regulators&diff=11728&oldid=prevMDolinar at 08:25, 14 November 20162016-11-14T08:25:09Z<p></p>
<table style="background-color: #fff; color: #202122;" data-mw="interface">
<col class="diff-marker" />
<col class="diff-content" />
<col class="diff-marker" />
<col class="diff-content" />
<tr class="diff-title" lang="en">
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 08:25, 14 November 2016</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l1">Line 1:</td>
<td colspan="2" class="diff-lineno">Line 1:</td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>[<del style="font-weight: bold; text-decoration: none;">http://</del>http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>[http://www.elowitz.caltech.edu/publications/Repressilator.pdf A synthetic oscillatory network of transcriptional regulators]</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>Michael B. Elowitz & Stanislas Leibler</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>Michael B. Elowitz & Stanislas Leibler</div></td></tr>
</table>MDolinarhttps://wiki.fkkt.uni-lj.si/index.php?title=A_synthetic_oscillatory_network_of_transcriptional_regulators&diff=9785&oldid=prevValterB at 09:55, 4 January 20152015-01-04T09:55:23Z<p></p>
<table style="background-color: #fff; color: #202122;" data-mw="interface">
<col class="diff-marker" />
<col class="diff-content" />
<col class="diff-marker" />
<col class="diff-content" />
<tr class="diff-title" lang="en">
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 09:55, 4 January 2015</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l48">Line 48:</td>
<td colspan="2" class="diff-lineno">Line 48:</td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Results ==</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Results ==</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour over 40 % of the observed cells were shown to follow such regime. Despite that the noise in individual cells due to stochastic effects coupled with inability of cells to synchronize oscillations across population caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk. Because of such effects the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at <del style="font-weight: bold; text-decoration: none;">30°C</del>) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>As mentioned previously, the authors have used intermediate stability variant of GFP, coupled to one of the promoters within the represillator, to quantitatively measure the state of the oscillatory system. This reporter system was introduced in a high copy plasmid in contrast to represillator system, which was encoded on a low-copy plasmid. Due to the modifications introduced to promote oscillatory behaviour<ins style="font-weight: bold; text-decoration: none;">, </ins>over 40 % of the observed cells were shown to follow such regime. Despite that<ins style="font-weight: bold; text-decoration: none;">, </ins>the noise in individual cells due to stochastic effects<ins style="font-weight: bold; text-decoration: none;">, </ins>coupled with inability of cells to synchronize oscillations across population<ins style="font-weight: bold; text-decoration: none;">, </ins>caused rapid desynchronisation and inability to measure fluorescence fluctuations in bulk<ins style="font-weight: bold; text-decoration: none;">. Attempts at synchronisation of cell population oscillators using IPTG were unsuccessful</ins>. Because of such effects<ins style="font-weight: bold; text-decoration: none;">, </ins>the measurements of represillator behavior must occur on individual-cell level and was done under a fluorescent microscope. Using it<ins style="font-weight: bold; text-decoration: none;">, </ins>the authors measured fluorescence of a large number of individual cells as a function of time. The oscillation period was shown to be on average longer than the replication period of cells; the measurements were therefore continued on daughter cells. Interestingly, the desynchronisation seems to be relatively slow in sister cells as shown in figures 3a-c, therefore we can assume that the replication process doesn’t interfere substantially with the oscillatory system. In such experiments, total observation time was limited to about 10 hours (at <ins style="font-weight: bold; text-decoration: none;">30 °C</ins>) due to colonies entering stationary phase. After entering stationary phase the represillator grinds to a halt, indicating its direct dependence on endogenous global regulatory mechanisms.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause <del style="font-weight: bold; text-decoration: none;">other </del>effects, such as <del style="font-weight: bold; text-decoration: none;">substantial </del>changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>The timecourse of the fluorescence of on cell harboring the represillator and reporter system is showed in figure 2 in the article. We can see that the amplitude of fluorescence oscillations is large compared to the background noise, with peak-to-peak intervals of 160±40 min (mean ± s.d.). Typical cell division time under used conditions was 50 – 70 min. As mentioned before, sibling decorrelation time was relatively long, measured at 95 ± 10 min. Cell duplication was observed to cause <ins style="font-weight: bold; text-decoration: none;">various </ins>effects, such as changes in oscillation frequency and amplitude as well as producing phase delay in one cell relative to the other. Interestingly, when the experiment was repeated using the same genetic constructs in the same strain of host cells under similar conditions, oscillation frequencies observed were remarkably different.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Theoretical work ==</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Theoretical work ==</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>At the end of the article authors contemplate the theoretical basis behind the work<del style="font-weight: bold; text-decoration: none;">, </del>described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation <del style="font-weight: bold; text-decoration: none;">in </del>natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance<del style="font-weight: bold; text-decoration: none;">; </del>the authors ask whether <del style="font-weight: bold; text-decoration: none;">construction of </del>similar circuits <del style="font-weight: bold; text-decoration: none;">that </del>would retain this highly valued property <del style="font-weight: bold; text-decoration: none;">is </del>possible <del style="font-weight: bold; text-decoration: none;">as a </del>logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>At the end of the article<ins style="font-weight: bold; text-decoration: none;">, </ins>authors contemplate the theoretical basis behind the work described in this article (box 1 in the article). They mention that the stochastic effects may be the culprit responsible for noisy operation <ins style="font-weight: bold; text-decoration: none;">of </ins>natural gene-expression networks as well as their own. We should be reminded at this stage that all events, chemical by nature, are inherently stochastic as well as of discreteness of the network components. Corpuscularity itself is sufficient to greatly increase noise if the number of involved molecules is low enough. They have conducted simulations that show those effects reduce the correlation time of individual cells to about two periods. As we know that natural circadian clocks exhibit great noise resistance<ins style="font-weight: bold; text-decoration: none;">, </ins>the authors ask whether similar <ins style="font-weight: bold; text-decoration: none;">synthetic </ins>circuits would retain this highly valued property <ins style="font-weight: bold; text-decoration: none;">and mention it as </ins>possible logical continuation of their work. Such network would use both positive and negative regulation elements which could lead to a formation of a bistable behavior with high noise-resistance.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
</table>ValterBhttps://wiki.fkkt.uni-lj.si/index.php?title=A_synthetic_oscillatory_network_of_transcriptional_regulators&diff=9784&oldid=prevValterB at 09:45, 4 January 20152015-01-04T09:45:46Z<p></p>
<table style="background-color: #fff; color: #202122;" data-mw="interface">
<col class="diff-marker" />
<col class="diff-content" />
<col class="diff-marker" />
<col class="diff-content" />
<tr class="diff-title" lang="en">
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 09:45, 4 January 2015</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l44">Line 44:</td>
<td colspan="2" class="diff-lineno">Line 44:</td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they combined λ P<sub>L</sub> promoter with lac and tet operator sequences producing hybrid versions, which are stronger yet still tightly repressible, termed P<sub>L</sub>lac01 and P<sub>L</sub>tet01 respectively (be reminded to consult figure 1a in article or the list above as required). The third promoter used is termed λP<sub>R</sub> and is naturally occurring right promoter of lambda phage; it already contains operator sites for the lambda cI protein that represses the promoter. Additionally, they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes - close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell. Low enough half-life of involved proteins is paramount for oscillations to be transferred to protein stage with little phase delay. If life-span of protein is short compared to the peak-to-peak time of oscillation, proteins synthesized at the time of the peak will clear from the cell quickly and therefore enhance the dynamics of the system by being in-phase with the transcriptional oscillations.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they combined λ P<sub>L</sub> promoter with lac and tet operator sequences producing hybrid versions, which are stronger yet still tightly repressible, termed P<sub>L</sub>lac01 and P<sub>L</sub>tet01 respectively (be reminded to consult figure 1a in article or the list above as required). The third promoter used is termed λP<sub>R</sub> and is naturally occurring right promoter of lambda phage; it already contains operator sites for the lambda cI protein that represses the promoter. Additionally, they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes - close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell. Low enough half-life of involved proteins is paramount for oscillations to be transferred to protein stage with little phase delay. If life-span of protein is short compared to the peak-to-peak time of oscillation, proteins synthesized at the time of the peak will clear from the cell quickly and therefore enhance the dynamics of the system by being in-phase with the transcriptional oscillations.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter P<sub>L</sub>tetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed <del style="font-weight: bold; text-decoration: none;">this, </del>so called reporter<del style="font-weight: bold; text-decoration: none;">, </del>construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter P<sub>L</sub>tetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed <ins style="font-weight: bold; text-decoration: none;">the </ins>so called reporter construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator <ins style="font-weight: bold; text-decoration: none;">system</ins>. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Results ==</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>== Results ==</div></td></tr>
</table>ValterBhttps://wiki.fkkt.uni-lj.si/index.php?title=A_synthetic_oscillatory_network_of_transcriptional_regulators&diff=9783&oldid=prevValterB at 09:43, 4 January 20152015-01-04T09:43:26Z<p></p>
<table style="background-color: #fff; color: #202122;" data-mw="interface">
<col class="diff-marker" />
<col class="diff-content" />
<col class="diff-marker" />
<col class="diff-content" />
<tr class="diff-title" lang="en">
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 09:43, 4 January 2015</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l42">Line 42:</td>
<td colspan="2" class="diff-lineno">Line 42:</td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they combined λ P<sub>L</sub> promoter with lac and tet operator sequences producing hybrid versions, which are stronger yet still tightly repressible, termed P<sub>L</sub>lac01 and P<sub>L</sub>tet01 respectively (be reminded to consult figure 1a in article or the list above as required). The third promoter used is termed λP<sub>R</sub> and is naturally occurring right promoter of lambda phage; it already contains operator sites for the lambda cI protein that represses the promoter. Additionally, they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes - close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they combined λ P<sub>L</sub> promoter with lac and tet operator sequences producing hybrid versions, which are stronger yet still tightly repressible, termed P<sub>L</sub>lac01 and P<sub>L</sub>tet01 respectively (be reminded to consult figure 1a in article or the list above as required). The third promoter used is termed λP<sub>R</sub> and is naturally occurring right promoter of lambda phage; it already contains operator sites for the lambda cI protein that represses the promoter. Additionally, they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes - close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell<ins style="font-weight: bold; text-decoration: none;">. Low enough half-life of involved proteins is paramount for oscillations to be transferred to protein stage with little phase delay. If life-span of protein is short compared to the peak-to-peak time of oscillation, proteins synthesized at the time of the peak will clear from the cell quickly and therefore enhance the dynamics of the system by being in-phase with the transcriptional oscillations</ins>.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter P<sub>L</sub>tetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter P<sub>L</sub>tetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.</div></td></tr>
</table>ValterBhttps://wiki.fkkt.uni-lj.si/index.php?title=A_synthetic_oscillatory_network_of_transcriptional_regulators&diff=9782&oldid=prevValterB at 09:34, 4 January 20152015-01-04T09:34:10Z<p></p>
<table style="background-color: #fff; color: #202122;" data-mw="interface">
<col class="diff-marker" />
<col class="diff-content" />
<col class="diff-marker" />
<col class="diff-content" />
<tr class="diff-title" lang="en">
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 09:34, 4 January 2015</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l42">Line 42:</td>
<td colspan="2" class="diff-lineno">Line 42:</td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they combined λ P<sub>L</sub> promoter with lac and tet operator sequences producing hybrid versions, which are stronger yet still tightly repressible, termed P<sub>L</sub>lac01 and P<sub>L</sub>tet01 respectively (be reminded to consult figure 1a in article or the list above as required). The third promoter used is termed λP<sub>R</sub> and is naturally occurring right promoter of lambda phage; it already contains operator sites for the lambda cI protein that represses the promoter. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes<del style="font-weight: bold; text-decoration: none;">, </del>close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they combined λ P<sub>L</sub> promoter with lac and tet operator sequences producing hybrid versions, which are stronger yet still tightly repressible, termed P<sub>L</sub>lac01 and P<sub>L</sub>tet01 respectively (be reminded to consult figure 1a in article or the list above as required). The third promoter used is termed λP<sub>R</sub> and is naturally occurring right promoter of lambda phage; it already contains operator sites for the lambda cI protein that represses the promoter. Additionally<ins style="font-weight: bold; text-decoration: none;">, </ins>they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes <ins style="font-weight: bold; text-decoration: none;">- </ins>close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter P<sub>L</sub>tetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter P<sub>L</sub>tetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.</div></td></tr>
</table>ValterBhttps://wiki.fkkt.uni-lj.si/index.php?title=A_synthetic_oscillatory_network_of_transcriptional_regulators&diff=9781&oldid=prevValterB at 09:32, 4 January 20152015-01-04T09:32:43Z<p></p>
<table style="background-color: #fff; color: #202122;" data-mw="interface">
<col class="diff-marker" />
<col class="diff-content" />
<col class="diff-marker" />
<col class="diff-content" />
<tr class="diff-title" lang="en">
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 09:32, 4 January 2015</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l42">Line 42:</td>
<td colspan="2" class="diff-lineno">Line 42:</td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they combined λ P<sub>L</sub> promoter with lac and tet operator sequences producing hybrid versions, which are stronger yet still tightly repressible, termed P<sub>L</sub>lac01 and P<sub>L</sub>tet01 respectively (be reminded to consult figure 1a in article or the list above as required). The third promoter used is termed <del style="font-weight: bold; text-decoration: none;">λPR </del>and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they combined λ P<sub>L</sub> promoter with lac and tet operator sequences producing hybrid versions, which are stronger yet still tightly repressible, termed P<sub>L</sub>lac01 and P<sub>L</sub>tet01 respectively (be reminded to consult figure 1a in article or the list above as required). The third promoter used is termed <ins style="font-weight: bold; text-decoration: none;">λP<sub>R</sub> </ins>and is naturally occurring right promoter of lambda phage<ins style="font-weight: bold; text-decoration: none;">; it already contains operator sites for the lambda cI protein that represses the promoter</ins>. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter P<sub>L</sub>tetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter P<sub>L</sub>tetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.</div></td></tr>
</table>ValterBhttps://wiki.fkkt.uni-lj.si/index.php?title=A_synthetic_oscillatory_network_of_transcriptional_regulators&diff=9780&oldid=prevValterB at 09:29, 4 January 20152015-01-04T09:29:32Z<p></p>
<table style="background-color: #fff; color: #202122;" data-mw="interface">
<col class="diff-marker" />
<col class="diff-content" />
<col class="diff-marker" />
<col class="diff-content" />
<tr class="diff-title" lang="en">
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 09:29, 4 January 2015</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l42">Line 42:</td>
<td colspan="2" class="diff-lineno">Line 42:</td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator. Reader can find more information on the subject by consulting figure 1b and 1c in the article.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they <del style="font-weight: bold; text-decoration: none;">used hybrid versions which are stronger yet still tightly repressible and combine </del>λ P<sub>L</sub> promoter with lac and tet operator sequences termed P<sub>L</sub>lac01 and P<sub>L</sub>tet01 respectively (be reminded to consult figure 1a in article or <del style="font-weight: bold; text-decoration: none;">figure I in this chapter if </del>required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they <ins style="font-weight: bold; text-decoration: none;">combined </ins>λ P<sub>L</sub> promoter with lac and tet operator sequences <ins style="font-weight: bold; text-decoration: none;">producing hybrid versions, which are stronger yet still tightly repressible, </ins>termed P<sub>L</sub>lac01 and P<sub>L</sub>tet01 respectively (be reminded to consult figure 1a in article or <ins style="font-weight: bold; text-decoration: none;">the list above as </ins>required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter P<sub>L</sub>tetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>At this stage the authors have constructed and characterized well-performing repressible promoters and repressors and combined all appropriate constructs onto a low copy-number plasmid to be transferred into ''E. coli''. There is yet still a most foundamental component missing – the way to see what is happening in the cell. The constructed circuit is expected to produce oscillations of all involved proteins’ concentrations, yet the technology to measure them in an easy and real-time fashion is missing. Therefore the authors coupled oscillations of TetR-lite repressor’s concentrations with expression of intermediate stability variant of green fluorescent protein (GFP) by putting the latter under TetR responsive promoter P<sub>L</sub>tetO1 (the same is used in the device itself to controll the expression of λ cI-lite repressor). They have constructed this, so called reporter, construct on a high copy number plasmid and used it to transfect ''E. coli'' cells already harboring the oscillator. Now the oscillations from the represillator are transferred to the reporter and can be easily observed under a fluorescent microscope as oscillations of fluorescence due to changing concentration of GFP in the cells.</div></td></tr>
</table>ValterBhttps://wiki.fkkt.uni-lj.si/index.php?title=A_synthetic_oscillatory_network_of_transcriptional_regulators&diff=9779&oldid=prevValterB at 09:24, 4 January 20152015-01-04T09:24:59Z<p></p>
<table style="background-color: #fff; color: #202122;" data-mw="interface">
<col class="diff-marker" />
<col class="diff-content" />
<col class="diff-marker" />
<col class="diff-content" />
<tr class="diff-title" lang="en">
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 09:24, 4 January 2015</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l40">Line 40:</td>
<td colspan="2" class="diff-lineno">Line 40:</td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>This list is a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. At this point reader is encouraged to fully understand the cyclical behaviour of the represillator, as the concept is a vital part of this chapter. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors. These phenomena are explained in more detail in the following paragraphs.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>This list is a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. At this point reader is encouraged to fully understand the cyclical behaviour of the represillator, as the concept is a vital part of this chapter. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors. These phenomena are explained in more detail in the following paragraphs.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state<ins style="font-weight: bold; text-decoration: none;">. Such properties move the biological system closer to a theoretical binary oscillator. In such a device, all components have only two valid output states, 0 or 1. In represillator terms, the expression of all repressor components would therefore be either maximal or zero. This would leave no room for a stable steady-state in a configuration using an odd number of components - like three, used in the represillator</ins>. Reader can find more information on the subject by consulting figure 1b and 1c in the article.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ P<sub>L</sub> promoter with lac and tet operator sequences termed P<sub>L</sub>lac01 and P<sub>L</sub>tet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>At the beginning, construction of a tripartite negative-feedback loop was envisioned using exclusively naturally occurring components – the so called wild-type forms. The authors’ estimates of relevant parameters of the system indicated that such configuration should by nature favour oscillatory regime and should not collapse to a steady-state. However, there are some issues that can easily be overcome by use of simple molecular manipulation methods, such as leakiness of lacI promoter and high half-lives of involved proteins compared to their RNA counterparts. They have chosen to increase their chances by adding several modifications to the natural components. Instead of wild-type versions of promoters they used hybrid versions which are stronger yet still tightly repressible and combine λ P<sub>L</sub> promoter with lac and tet operator sequences termed P<sub>L</sub>lac01 and P<sub>L</sub>tet01 respectively (be reminded to consult figure 1a in article or figure I in this chapter if required). The third promoter used is termed λPR and is naturally occurring right promoter of lambda phage. Additionally they lowered the repressor protein lifetimes to values closer to expected mRNA lifetimes (about 2 min on average in ''E. coli'') by inserting carboxy-terminal tag originating from ssrA RNA, which is recognized by host’s proteases and targets the protein for recycling. Data from previous experiments indicates that they can expect such tag to reduce the half-lives of repressor proteins to an order of magnitude of minutes, close to expected half-lives of mRNA. Repressors tagged in such manner were denoted LacI-, TetR- and λ cl-lite repressors due to their shortened life span in the cell.</div></td></tr>
</table>ValterBhttps://wiki.fkkt.uni-lj.si/index.php?title=A_synthetic_oscillatory_network_of_transcriptional_regulators&diff=9778&oldid=prevValterB at 09:18, 4 January 20152015-01-04T09:18:45Z<p></p>
<table style="background-color: #fff; color: #202122;" data-mw="interface">
<col class="diff-marker" />
<col class="diff-content" />
<col class="diff-marker" />
<col class="diff-content" />
<tr class="diff-title" lang="en">
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 09:18, 4 January 2015</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l38">Line 38:</td>
<td colspan="2" class="diff-lineno">Line 38:</td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>• promoter (P<sub>L</sub>tetO1, repressed by TetR) - λ cI-lite (repressor)</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>• promoter (P<sub>L</sub>tetO1, repressed by TetR) - λ cI-lite (repressor)</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>This list is a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>This list is a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop<ins style="font-weight: bold; text-decoration: none;">. At this point reader is encouraged to fully understand the cyclical behaviour of the represillator, as the concept is a vital part of this chapter</ins>. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors<ins style="font-weight: bold; text-decoration: none;">. These phenomena are explained in more detail in the following paragraphs</ins>.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.</div></td></tr>
</table>ValterBhttps://wiki.fkkt.uni-lj.si/index.php?title=A_synthetic_oscillatory_network_of_transcriptional_regulators&diff=9777&oldid=prevValterB at 09:14, 4 January 20152015-01-04T09:14:59Z<p></p>
<table style="background-color: #fff; color: #202122;" data-mw="interface">
<col class="diff-marker" />
<col class="diff-content" />
<col class="diff-marker" />
<col class="diff-content" />
<tr class="diff-title" lang="en">
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">← Older revision</td>
<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 09:14, 4 January 2015</td>
</tr><tr><td colspan="2" class="diff-lineno" id="mw-diff-left-l38">Line 38:</td>
<td colspan="2" class="diff-lineno">Line 38:</td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>• promoter (P<sub>L</sub>tetO1, repressed by TetR) - λ cI-lite (repressor)</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>• promoter (P<sub>L</sub>tetO1, repressed by TetR) - λ cI-lite (repressor)</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>This list <del style="font-weight: bold; text-decoration: none;">represents </del>a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div>This list <ins style="font-weight: bold; text-decoration: none;">is </ins>a simplified representation of a represillator. Each component represses the expression of the next component in line and the last one represses the first one closing the circle. This, under certain circumstances, produces temporal oscillations of components’ protein concentrations. The first repressor protein LacI inhibits the formation of second repressor protein TetR at the stage of transcription. In a similar manner expression of λ cI gene from λ phage is repressed by TetR repressor. The λ cI repressor closes the circle by repressing expression of LacI repressor protein thus forming a cyclical negative-feedback loop. Such system configuration has at least two types of solutions: convergence towards a stable steady state or an unstable steady state leading to sustained temporal oscilations. The system will fall into its native state immediately after introduction of transgenic material into host cell without exogenic interference. The authors have tried to use IPTG as an inhibitor of activity of LacI to synchronize states of all represillators in population but the results were marked by rapid decorrelation of states due to endogenous factors.</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>There are some prerequisites for such a system to behave as an oscillator and not sink into a stable stationary, or non-oscillatory, state. The authors have found that the oscillatory behavior is favored by strong promoters together with efficient ribosome-binding sites, which together produces a large amount of protein product in short amount of time. Tight repression, meaning low ‘leakiness’ of the promoters in repressed state, also contributes to such behavior as it increases the difference in protein concentrations in repressed vs. active state. Cooperative repression characteristics as well as comparable mRNA and protein degradation times are also beneficial to the prevalence of an oscillatory state. Reader can find more information on the subject by consulting figure 1b and 1c in the article.</div></td></tr>
</table>ValterB