Difference between revisions of "Team:Marburg/test joana"

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         B A S I C<span style="visibility: hidden;">A</span>P A R T S
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         C O M P O S I T E<span style="visibility: hidden;">A</span>P A R T S
 
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       <section class="section">
 
       <section class="section">
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         <h1 class="title">The origin</h1>
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         <h1 class="title">Composite Parts – level 2 Terminators</h1>
 
         <p style="text-align: justify;">
 
         <p style="text-align: justify;">
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                 Inspired by the fast progress in synthetic biology and its urgent need for genetic tools that enable the
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                 <div>  
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      exploitation of cyanobacteria for research and biotechnological applications, we set out to construct the most
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                        <p>Transcriptional terminators play a key role in regulating natural and synthetic genetic systems <a href="https://www.ncbi.nlm.nih.gov/pubmed/23511967">(Cambray et al., 2013)</a>. Therefore, it is valuable to know how efficient such a terminator is. The termination efficiency can be detected by measurement. In order to do so a dual fluorescent protein is used <a href="https://parts.igem.org/Help:Terminators/Measurement ">(“Help: Terminators/Measurement”)</a>. This means, there are two reporters in a plasmid and the fluorescence ratio of these two reporters is going to be compared to detect the termination efficiency. The terminator that is going to be measured is in the middle of these two reporters.</p>
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      versatile shuttle vector for cyanobacteria based on the modular Golden Gate Assembly method, allowing for flexible
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                        <p>In the absence of a terminator, the fluorescence ratio is normalized to 1 which is one calibration of this measurement <a href="https://parts.igem.org/Help:Terminators/Measurement ">(“Help: Terminators/Measurement”)</a>. Another method to calibrate this measurement is to measure the reporters individually, so having the same compounds on the plasmid, just changing the reporter <a href="https://core.ac.uk/download/pdf/4410463.pdf ">(Haiyao Huang, 2008)</a>. Furthermore, the measurement can be calibrated by switching the fluorescing parts to measure input and output of each of them  <a href="https://core.ac.uk/download/pdf/4410463.pdf ">(Haiyao Huang, 2008)</a>. </p>
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      cloning into a reliable self-replicating system.<br>
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                        <p>For the measurement the terminator is flanked by two reporters e.g. a GFP preceding the terminator that shall be measured and a RFP following the terminator  <a href="https://core.ac.uk/download/pdf/4410463.pdf ">(Haiyao Huang, 2008)</a>. The fluorescence ratio of the two reporters is then compared, revealing the termination efficiency. A terminator with a 100% termination efficiency should result in no fluorescence from the second reporter so in this example mentioned above no red signal should be seen  <a href="https://core.ac.uk/download/pdf/4410463.pdf ">(Haiyao Huang, 2008)</a>.</p>
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                <br>
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                        <p>Following the same principle, the efficiency of our level 2 terminators is measured. The terminator that shall be measured is flanked by two reporters. We used a m.Turquoise as preceding reporter and a YFP as following reporter. By comparing the fluorescence ratio of these two reporters, we are able to detect the termination efficiency.</p>
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            </p>
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                        </div>
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            <figure>
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                    <img style="display: block; margin: 0 auto 0 auto; width:50%"
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                        </div>
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                      src="https://lh3.googleusercontent.com/8ko4suiu3NQ_qmRIeZf1k1sg5EUw8g4JXfkGG3xAmRk1dxaVlZQbzC9Uz-6ToGKXaAf5p_yx9MVHhlO3QdMmG_l0ukJ0OVQOWBzcouM-HOTc_ta7LblxiVtTdLKrf9q4bpzP6ZRP"
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                        <figure style="text-align:center">
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                      alt="Lvl1 ori">
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                              <img style="height: 320px; width: 1200px;"
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                    <figcaption>
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                                  src="https://static.igem.org/mediawiki/2019/f/f6/T--Marburg--Toolbox_TerminatormeasurementB.svg" alt="Toolbox Terminator Measurement B">
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                      Fig.1 - Lvl1 ori
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                              <figcaption style="max-width: 2400px; text-align: center">
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                    </figcaption>
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                                  Fig.1 - Measurement of a level 2 terminator.
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                  </figure>
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                              </figcaption>
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                          </figure>
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            <div><p>
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                    Introduction of exogenous DNA can be done in multiple ways and propagated in a strain if it is integrated in the
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                        </body>
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                    chromosome or stably expressed on a self-replicating plasmid.<br> For rapid prototyping in cyanobacteria
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                    self-replicating plasmids are of higher interest than genome-integrations, as the latter can be quite
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                    time-consuming in cyanobacterial strains with multiple genome copies (<a
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                      href="https://www.ncbi.nlm.nih.gov/pubmed/22092711">Griese <i>et al.,</i> 2011</a>). Furthermore, genes
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                    introduced in self-replicating vectors have been shown to have higher gene-expression levels than those integrated
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                    in the genome, as copy numbers are typically higher (<a href="https://doi.org/10.1099/mic.0.000377">Chen Titel anhand dieser DOI in Citavi-Projekt übernehmen <i>et
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                        al.,</i> 2016</a>) – a desirable trait, not just for rapid prototyping in research applications, but also for
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                    biotechnological production of valuable compounds.<br>
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                    With our shuttle-vectors encompass a cyanobacterial origin of replication (ori) from <i>Synechococcus
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                      elongatus</i> PCC7942 as well as an <i>E.coli</i> ori, which is perfect for fast cloning processes, as these
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                    vectors can be easily recovered from the cyanobacteria and reintroduced in an <i>E.coli</i> strain.<br>
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                <br>
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            </p>
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            <br>
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            <p style="font-size: 20px">
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              Currently existing shuttle vectors for cyanobacteria are still based on standard systems working with multiple
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              cloning sites (MCS) for expression of homologous genes (<a href="https://doi.org/10.1099/mic.0.000377">Chen Titel anhand dieser DOI in Citavi-Projekt übernehmen <i>et
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                  al.,</i> 2016</a>). A huge downside is that these vectors include either an MCS (e.g. pAM5188) or a
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              fluorescence reporter (e.g. pAM4787), which is unpractical for easy selection of recombinant clones. Additionally,
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              an MCS comes with possible sequence constraints due to restriction sites leaving unwanted base pairs in your
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              constructs.<br>
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              Facilitating and standardizing the process of engineering biological systems is one of the fundamental goals of
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              synthetic biology (<a href="https://doi.org/10.1186/1754-1611-2-5">Shetty Titel anhand dieser DOI in Citavi-Projekt übernehmen <i>et al.,</i> 2008</a>), so the
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              construction of a shuttle-vector based on a modular cloning method significantly improves the genetic toolbox we
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              created for genetic engineering and synthetic biology approaches in <i>S.elongatus</i> and other
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              cyanobacteria.<br>
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            </p>
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−
            <br>
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            <p style="font-size: 20px">
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              The commonly used <i>S.elongatus</i> strain PCC7942 carries two endogenous plasmids, the 46,4kb pANL (<a
+
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                href="https://www.ncbi.nlm.nih.gov/pubmed/18353436">Chen <i>et al.,</i> 2008</a>) which is essential and the
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              7,8kb pANS (<a href="https://www.ncbi.nlm.nih.gov/pubmed/1552863">Van der Plas <i>et al.,</i> 1992</a>) which is
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              not essential for the strain and can easily be cured.<br>
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              This small plasmid has already been used for construction of shuttle vectors (<a
+
−
                href="https://doi.org/10.1016/0076-6879(87)53054-3">Kuhlemeier Titel anhand dieser DOI in Citavi-Projekt übernehmen & van Arkel, 1987</a> ; <a
+
−
                href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC217787/">Golden & Sherman, 1983</a> ; <a
+
−
                href="https://doi.org/10.1099/mic.0.000377">Chen Titel anhand dieser DOI in Citavi-Projekt übernehmen <i>et al.,</i> 2016</a>). <br>
+
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              We followed this lead to create the best shuttle-vector available for cyanobacteria by encompassing the minimal
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              replication region of pANS and the ColE1 origin of replication into our vectors, allowing for stable
+
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              self-replication with high copy numbers in cyanobacteria (<a href="https://doi.org/10.1099/mic.0.000377">Chen Titel anhand dieser DOI in Citavi-Projekt übernehmen
+
−
                <i>et al.,</i> 2016</a>) and <i>E.coli</i> (<a href="https://doi.org/10.1016/S0065-2660(02)46013-0">Gerhart Titel anhand dieser DOI in Citavi-Projekt übernehmen
+
−
                <i>et al.,</i>2002</a>). This addition to the genetic toolbox proves invaluable, as it can be easily recovered
+
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              from the cyanobacterial strain and reintroduced in <i>E.coli</i> for fast GoldenGate-based cloning processes.<br>
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            </p>
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            <br>
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            <p style="font-size: 20px">
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              In order to supply the community with an easy selection system, we equipped our shuttle vector with a fluorescent
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              reporter that is cut out when introducing new genetic parts:<br>
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              A mRFP (red fluorescent protein) cassette is flanked by our standardized TypeIIS restriction enzyme recognition
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              sequences (BsmBI or BsaI depending on what level you want to clone in). In a standard Golden Gate reaction this
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              cassette will drop out and leave space for the parts that should be introduced, allowing for easy selection on
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              plate after successful cloning – red colonies are wrong, still harboring the mRFP cassette and white colonies (if
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              no other fluorescence is introduced) are correct, as the mRFP was switched with the parts of interest.<br>
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              <br>
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              This crucial part comes in two variations - one for cloning Lvl1 and one for Lvl2 constructs -, giving the Golden
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              Gate community everything they need for successful and reliable creation of self-replicating vectors in
+
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              cyanobacteria.
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            </p>
+
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            <br>
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            </p>
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          </div>
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          <br>
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         </main>
  
 
</html>
 
</html>

Revision as of 23:14, 21 October 2019

C O M P O S I T EAP A R T S

Composite Parts – level 2 Terminators

Transcriptional terminators play a key role in regulating natural and synthetic genetic systems (Cambray et al., 2013). Therefore, it is valuable to know how efficient such a terminator is. The termination efficiency can be detected by measurement. In order to do so a dual fluorescent protein is used (“Help: Terminators/Measurement”). This means, there are two reporters in a plasmid and the fluorescence ratio of these two reporters is going to be compared to detect the termination efficiency. The terminator that is going to be measured is in the middle of these two reporters.

In the absence of a terminator, the fluorescence ratio is normalized to 1 which is one calibration of this measurement (“Help: Terminators/Measurement”). Another method to calibrate this measurement is to measure the reporters individually, so having the same compounds on the plasmid, just changing the reporter (Haiyao Huang, 2008). Furthermore, the measurement can be calibrated by switching the fluorescing parts to measure input and output of each of them (Haiyao Huang, 2008).

For the measurement the terminator is flanked by two reporters e.g. a GFP preceding the terminator that shall be measured and a RFP following the terminator (Haiyao Huang, 2008). The fluorescence ratio of the two reporters is then compared, revealing the termination efficiency. A terminator with a 100% termination efficiency should result in no fluorescence from the second reporter so in this example mentioned above no red signal should be seen (Haiyao Huang, 2008).

Following the same principle, the efficiency of our level 2 terminators is measured. The terminator that shall be measured is flanked by two reporters. We used a m.Turquoise as preceding reporter and a YFP as following reporter. By comparing the fluorescence ratio of these two reporters, we are able to detect the termination efficiency.

Toolbox Terminator Measurement B
Fig.1 - Measurement of a level 2 terminator.