Difference between revisions of "Part:BBa K3228069"

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  <h4>Cyanobacterial shuttle vectors </h4>
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                    As we have already clarified in the description part, self replicating shuttle vectors are essential for many
      <hr class="line">
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                    workflows, as the gene expression levels are higher and non of the tedious selection processes that come with
 
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                    genomic integrations have to be done. <br>
    </div>
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    <div style="margin-top: 10vh;">
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                    On our road to the modular vector we were seeking, we firstly cured our own S. elongatus UTEX 2973 strain of its
      <section class="section">
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                    pANS plasmid. This was done by transforming the pAM4787 vector, which holds a spectinomycin resistance as well as a
        <h1 class="title">The origin</h1>
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                    YFP cassette
        <p style="text-align: justify;">
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                    <a href= https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000377> (Chen et al., 2016)</a>.
                Inspired by the fast progress in Synthetic Biology and its urgent need for genetic tools that enable the
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                    Due to plasmid incompatibility - explained here in our design section <b>[Link to shuttle vector design]</b> - and because antibiotic pressure is applied, the pANS plasmid was over time cured from the
      exploitation of cyanobacteria for research and biotechnological applications, we set out to construct the most
+
                    strain, which then just kept the pAM4787 plasmid. Transformation was done by conjugation with the pRK2013 plasmid in
      versatile shuttle vector for cyanobacteria based on the modular Golden Gate Assembly method, allowing for flexible
+
                    DH5ɑ and the pAM4787 in HB101. Both were grown to an OD600≈0.5, washed in LB and mixed with S. elongatus which was
      cloning into a reliable self-replicating system.<br>
+
                    grown to late exponential phase and then washed in BG11.
                <br>
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                    We could clearly show, that the conjugant strain bears the pAM4787 plasmid if selective pressure is held up.
            </p>
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                    <figure Style="text-align:center">
            <figure>
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                            <img style="height: 65ex; width: 50ex"
                    <img style="display: block; margin: 0 auto 0 auto; width:50%"
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                                src= https://2019.igem.org/wiki/images/e/eb/T--Marburg--YFPconstructConjugantFACS.png
                      src="https://lh3.googleusercontent.com/8ko4suiu3NQ_qmRIeZf1k1sg5EUw8g4JXfkGG3xAmRk1dxaVlZQbzC9Uz-6ToGKXaAf5p_yx9MVHhlO3QdMmG_l0ukJ0OVQOWBzcouM-HOTc_ta7LblxiVtTdLKrf9q4bpzP6ZRP"
+
                                alt=" https://2019.igem.org/wiki/images/e/eb/T--Marburg--YFPconstructConjugantFACS.png">
                      alt="Lvl1 ori">
+
                            <figcaption>
                    <figcaption>
+
                                Fig. 11:  Cell counts of conjugant strain. The y axis shows relative YFP fluorescence and the x axis relative autofluorescence.
                      Fig.1 - Lvl1 ori
+
                            </figcaption>
                    </figcaption>
+
                        </figure>
                  </figure>
+
                    This was followed by us starting to culture the pAM4787 bearing strain without
           
+
                    antibiotics again, slowly removing selective pressure from the cells. As the plasmid does not give them any other
            <div><p>
+
                     advantage and is probably just more metabolic burden due to the constantly produced YFP proteins it is slowly being
                     Introduction of exogenous DNA can be done in multiple ways and propagated in a strain if it is integrated in the
+
                    lost.
                     chromosome or stably expressed on a self-replicating plasmid.<br> For rapid prototyping in cyanobacteria
+
                    We could prove this in multiple setups: with the flow cytometry device we were kindly granted access to we could
                     self-replicating plasmids are of higher interest than genome-integrations, as the latter can be quite
+
                     clearly show the missing YFP signal in the cured <i>S. elongatus </i>strain  and
                     time-consuming in cyanobacterial strains with multiple genome copies (<a
+
                     logically this could also be observed over our UV table
                      href="https://www.ncbi.nlm.nih.gov/pubmed/22092711">Griese <i>et al.,</i> 2011</a>). Furthermore, genes
+
                   
                    introduced in self-replicating vectors have been shown to have higher gene-expression levels than those integrated
+
                     <figure Style="text-align:center">
                    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
+
                            <img style="height: 65ex; width: 50ex"
                        al.,</i> 2016</a>) – a desirable trait, not just for rapid prototyping in research applications, but also for
+
                                src= https://2019.igem.org/wiki/images/c/c4/T--Marburg--CuredStrainsFACS.png
                    biotechnological production of valuable compounds.<br>
+
                                alt="https://2019.igem.org/wiki/images/c/c4/T--Marburg--CuredStrainsFACS.png ">
                    With our shuttle-vectors encompass a cyanobacterial origin of replication (ori) from <i>Synechococcus
+
                                <img style="height: 65ex; width: 50ex"
                      elongatus</i> PCC7942 as well as an <i>E.coli</i> ori, which is perfect for fast cloning processes, as these
+
                                src=  https://2019.igem.org/wiki/images/d/da/T--Marburg--CuredStrainUV.png
                     vectors can be easily recovered from the cyanobacteria and reintroduced in an <i>E.coli</i> strain.<br>
+
                                alt="https://2019.igem.org/wiki/images/d/da/T--Marburg--CuredStrainUV.png">
                <br>
+
                                <figcaption>
            </p>
+
                                    a) Cell counts of cured strain. The y axis shows relative YFP fluorescence and the x axis relative autofluorescence
            <br>
+
                                    b) Comparison of the fluorescence signal of the transformed (left) and cured (right) strain.
            <p style="font-size: 20px">
+
                                </figcaption> </figure>
              Currently existing shuttle vectors for cyanobacteria are still based on standard systems working with multiple
+
               
              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
+
                 
                  al.,</i> 2016</a>). A huge downside is that these vectors include either an MCS (e.g. pAM5188) or a
+
                 
              fluorescence reporter (e.g. pAM4787), which is unpractical for easy selection of recombinant clones. Additionally,
+
                     Furthermore we performed colony PCRs as a test. We sent our plasmid-free strain to Next Generation Sequencing in order to ensure that the strain really has lost the
              an MCS comes with possible sequence constraints due to restriction sites leaving unwanted base pairs in your
+
                    pANS plasmid.
              constructs.<br>
+
                    <figure Style="text-align:center">
              Facilitating and standardizing the process of engineering biological systems is one of the fundamental goals of
+
                            <img style="height: 65ex; width: 50ex"
              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
+
                                src=   https://2019.igem.org/wiki/images/e/e9/T--Marburg--ColonyPCRcuredStrain.jpg
              construction of a shuttle-vector based on a modular cloning method significantly improves the genetic toolbox we
+
                                alt="gel pcr">
              created for genetic engineering and synthetic biology approaches in <i>S.elongatus</i> and other
+
                            <figcaption> Fig. 12:  Colony PCR of the wild type, the conjugated and the cured strain.
              cyanobacteria.<br>
+
                          </figcaption>
            </p>
+
                        </figure>
            <br>
+
               
            <p style="font-size: 20px">
+
                    Our next step was the characterization of the cyanobacterial shuttle vector mentioned in our design section <b>[Link to
              The commonly used <i>S.elongatus</i> strain PCC7942 carries two endogenous plasmids, the 46,4kb pANL (<a
+
                    design]</b>.
                href="https://www.ncbi.nlm.nih.gov/pubmed/18353436">Chen <i>et al.,</i> 2008</a>) which is essential and the
+
                    In an extensive flow cytometry experiment we assessed the fluorescence of a transformed YFP-construct in our cured
              7,8kb pANS (<a href="https://www.ncbi.nlm.nih.gov/pubmed/1552863">Van der Plas <i>et al.,</i> 1992</a>) which is
+
                    strain, showing that the shuttle vector with the minimal replication element can be maintained in<i>S. elongatus </i> UTEX
              not essential for the strain and can easily be cured.<br>
+
                    2973 .
              This small plasmid has already been used for construction of shuttle vectors (<a
+
                    <figure Style="text-align:center">
                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
+
                            <img style="height: 65ex; width: 50ex"
                href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC217787/">Golden & Sherman, 1983</a> ; <a
+
                                src=   
                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>
+
                                alt="hier kommen noch 3 bilder von FACS Messung aber no time">
              We followed this lead to create the best shuttle-vector available for cyanobacteria by encompassing the minimal
+
                            <figcaption> Fig. 13:  hier kommen noch 3 bilder von FACS Messung aber no time.
              replication region of pANS and the ColE1 origin of replication into our vectors, allowing for stable
+
                          </figcaption>
              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
+
                        </figure>
                <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
+
                    After another four weeks of cultivation we looked at our
                <i>et al.,</i>2002</a>). This addition to the genetic toolbox proves invaluable, as it can be easily recovered
+
                    cultures again on the UV table to check if fluorescence was still present and the high intensity of the fluorescence
              from the cyanobacterial strain and reintroduced in <i>E.coli</i> for fast GoldenGate-based cloning processes.<br>
+
                    proved to us, that the plasmid is still stably replicated in our strain, showing us, that the minimal replication
            </p>
+
                    element does indeed work in our strain.
            <br>
+
                    For further analysis we performed qPCR with this transformed strain, in order to check the copy number of the
            <p style="font-size: 20px">
+
                    vector. We used the copy number of pANL as a reference, which is supposedly at ~2,6 copies per chromosome
              In order to supply the community with an easy selection system, we equipped our shuttle vector with a fluorescent
+
                    <a href= https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000377> (Chen et al., 2016)</a>. Our data shows a ~4,5 times higher copy number relative to pANL, meaning that the construct is
              reporter that is cut out when introducing new genetic parts:<br>
+
                    maintained with approximately 11,7 copies per chromosome.
              A mRFP (red fluorescent protein) cassette is flanked by our standardized TypeIIS restriction enzyme recognition
+
                    <figure Style="text-align:center">
              sequences (BsmBI or BsaI depending on what level you want to clone in). In a standard Golden Gate reaction this
+
                            <img style="height: 65ex; width: 50ex"
              cassette will drop out and leave space for the parts that should be introduced, allowing for easy selection on
+
                                src=   
              plate after successful cloning – red colonies are wrong, still harboring the mRFP cassette and white colonies (if
+
                                alt="hier kommen noch 3 bilder [FigXX Honrok qPCR data? diagramm biddeeeeeeeeeeeeeee]  aber no time">
              no other fluorescence is introduced) are correct, as the mRFP was switched with the parts of interest.<br>
+
                            <figcaption> Fig. 14:  hier kommen noch 3 bilder [FigXX Honrok qPCR data? diagramm biddeeeeeeeeeeeeeee] aber no time.
              <br>
+
                          </figcaption>
              This crucial part comes in two variations - one for cloning Lvl1 and one for Lvl2 constructs -, giving the Golden
+
                        </figure>
              Gate community everything they need for successful and reliable creation of self-replicating vectors in
+
                   
              cyanobacteria.
+
               
            </p>
+
                    Additionally we measured the fluorescence signals in a plate reader at different optical densities and could again
            <br>
+
                    confirm high fluorescence signals, indicating strong gene expression in constructs built around this replication
            </p>
+
                    element.
          </div>
+
                    <figure Style="text-align:center">
          <br>
+
                            <img style="height: 65ex; width: 50ex"
        </main>
+
                                src=  https://2019.igem.org/wiki/images/f/f0/T--Marburg--results_yfp_pam_4787_6_replicates.jpg 
 
+
                                alt="diagramm">
 +
                            <figcaption> Fig. 15:YFP fluorescence at different optical densities. </figcaption>
 +
                        </figure>
 +
                   
 +
                   
 +
                    All this data confirms that the construct actually works and can be reliably used as a cyanobacterial shuttle
 +
                    vector, proving that BBa_K3228069 works as intended, thus functioning as our validated part.
 +
                    This assumption is solidified by all our sequence data, showing that the shuttle vectors were completely assembled
 +
                    as planned in our design section <b>[Link to design of shuttle vectors]</b>  
 +
                    .
 +
                    <figure Style="text-align:center">
 +
                            <img style="height: 65ex; width: 50ex"
 +
                                src=  xyz 
 +
                                alt="disgramm">
 +
                            <figcaption> Fig. 16: [FigXX seq results of lvl1 and lvl2 ori] </figcaption>
 +
                        </figure>
 +
                    </p>
 +
       
 +
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Revision as of 01:49, 22 October 2019


pMC_0_7+8_panS_specResLVL1

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1199
    Illegal PstI site found at 3734
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 3734
    Illegal NotI site found at 1
    Illegal NotI site found at 5738
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4897
    Illegal XhoI site found at 1249
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1199
    Illegal PstI site found at 3734
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1199
    Illegal PstI site found at 3734
    Illegal NgoMIV site found at 48
    Illegal NgoMIV site found at 190
  • 1000
    COMPATIBLE WITH RFC[1000]

B A S I C P A R T S

Cyanobacterial shuttle vectors

As we have already clarified in the description part, self replicating shuttle vectors are essential for many workflows, as the gene expression levels are higher and non of the tedious selection processes that come with genomic integrations have to be done.
On our road to the modular vector we were seeking, we firstly cured our own S. elongatus UTEX 2973 strain of its pANS plasmid. This was done by transforming the pAM4787 vector, which holds a spectinomycin resistance as well as a YFP cassette (Chen et al., 2016). Due to plasmid incompatibility - explained here in our design section [Link to shuttle vector design] - and because antibiotic pressure is applied, the pANS plasmid was over time cured from the strain, which then just kept the pAM4787 plasmid. Transformation was done by conjugation with the pRK2013 plasmid in DH5ɑ and the pAM4787 in HB101. Both were grown to an OD600≈0.5, washed in LB and mixed with S. elongatus which was grown to late exponential phase and then washed in BG11. We could clearly show, that the conjugant strain bears the pAM4787 plasmid if selective pressure is held up.
 https://2019.igem.org/wiki/images/e/eb/T--Marburg--YFPconstructConjugantFACS.png
Fig. 11: Cell counts of conjugant strain. The y axis shows relative YFP fluorescence and the x axis relative autofluorescence.
This was followed by us starting to culture the pAM4787 bearing strain without antibiotics again, slowly removing selective pressure from the cells. As the plasmid does not give them any other advantage and is probably just more metabolic burden due to the constantly produced YFP proteins it is slowly being lost. We could prove this in multiple setups: with the flow cytometry device we were kindly granted access to we could clearly show the missing YFP signal in the cured S. elongatus strain and logically this could also be observed over our UV table
https://2019.igem.org/wiki/images/c/c4/T--Marburg--CuredStrainsFACS.png https://2019.igem.org/wiki/images/d/da/T--Marburg--CuredStrainUV.png
a) Cell counts of cured strain. The y axis shows relative YFP fluorescence and the x axis relative autofluorescence b) Comparison of the fluorescence signal of the transformed (left) and cured (right) strain.
Furthermore we performed colony PCRs as a test. We sent our plasmid-free strain to Next Generation Sequencing in order to ensure that the strain really has lost the pANS plasmid.
gel pcr
Fig. 12: Colony PCR of the wild type, the conjugated and the cured strain.
Our next step was the characterization of the cyanobacterial shuttle vector mentioned in our design section [Link to design]. In an extensive flow cytometry experiment we assessed the fluorescence of a transformed YFP-construct in our cured strain, showing that the shuttle vector with the minimal replication element can be maintained inS. elongatus UTEX 2973 .
Fig. 13: hier kommen noch 3 bilder von FACS Messung aber no time.
After another four weeks of cultivation we looked at our cultures again on the UV table to check if fluorescence was still present and the high intensity of the fluorescence proved to us, that the plasmid is still stably replicated in our strain, showing us, that the minimal replication element does indeed work in our strain. For further analysis we performed qPCR with this transformed strain, in order to check the copy number of the vector. We used the copy number of pANL as a reference, which is supposedly at ~2,6 copies per chromosome (Chen et al., 2016). Our data shows a ~4,5 times higher copy number relative to pANL, meaning that the construct is maintained with approximately 11,7 copies per chromosome.
Fig. 14: hier kommen noch 3 bilder [FigXX Honrok qPCR data? diagramm biddeeeeeeeeeeeeeee] aber no time.
Additionally we measured the fluorescence signals in a plate reader at different optical densities and could again confirm high fluorescence signals, indicating strong gene expression in constructs built around this replication element.
diagramm
Fig. 15:YFP fluorescence at different optical densities.
All this data confirms that the construct actually works and can be reliably used as a cyanobacterial shuttle vector, proving that BBa_K3228069 works as intended, thus functioning as our validated part. This assumption is solidified by all our sequence data, showing that the shuttle vectors were completely assembled as planned in our design section [Link to design of shuttle vectors] .
disgramm
Fig. 16: [FigXX seq results of lvl1 and lvl2 ori]

This part is contained in the Green Expansion, a range of parts from iGEM Marburg 2019that enables users of the Marburg Collection 2.0 to design MoClo compatible vectors for cyanobacteria as well as to engineer the genome of several cyanobacterial species.

The Green Expansion

The Green Expansion is an addition of parts to the Marburg Collection 2.0 (See: Design of the Marburg Collection) that features the world's first MoClo compatible shuttle vector for cyanobacteria. BBa_K3228069

Figure 1: Design of the first MoClo compatible shuttle vector for cyanobacteria for LVL 1 constructs. This can be used for the integration of simple genetic modules.


Figure 2: Design of the first MoClo compatible shuttle vector for cyanobacteria for LVL 2 constructs. This can be used for the integration of complex genetic devices.

The Green Expansion also offers all the parts needed for the genomic integration of one or multiple genes in cyanobacteria. This M.E.G.A. (Modularized Engineering of Genome Areas) kit convinces with a striking flexibility and a very intuitive workflow for the de novo assembly of your plasmid of choice. It encompasses five different neutral integration sites to choose from: three conventional sites frequently used in the cyanobacterial community (NSI to NSIII) as well as our own rationally designed artificial Neutral integration Site options a.N.S.o. 1 and 2 (See: Finding new artificial Neutral integration Site options).These sites show no transcriptional activity from neighboring regions according to RNA-seq data and are therefore completely orthogonal. Additionally we offer four different antibiotic markers to use (chloramphenicol, gentamicin, spectinomycin and kanamycin). With the Green Expansion up to 20 genes can be introduced into a cyanobacterial strain.

Figure 3: Overview over the modularized editing of genome area kit (M.E.G.A. kit)



Thanks to the flexible design this expansion can also be used for the genomic modification of any chassis after the introduction of a new species specific LVL 0 integration sites to our Marburg Collection 2.0. As the workflow to build new homologies is a bit more intricate compared to the one pot on step assembly of our other parts due to the internal BsmBI cutting site, we described the workflow for that in our design section (See: Design of neutral integration sites).

The Green Expansion proves a valuable addition to our Marburg Collection 2.0 and to the iGEM Registry of Standard Biological Parts. It services users of our chassis and other cyanobacterial strains with a useful tool for genomic modifications but it also contributes a shell that can be used to modify any other model organism as well.

Compability

These parts are compatible with the RCF [1000] standard and can be used in any part collection that uses the PhytoBrick standard of overhangs. For more information we recommend to head over to Design of the Marburg Collection iGEM Marburg 2018.

Parts of the Green Expansion