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<div class="box-dark"> | <div class="box-dark"> | ||
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alt="Syntex Logo"> | alt="Syntex Logo"> | ||
</div> | </div> | ||
− | < | + | <div style="margin-top: 11vh; "> |
+ | <section class="section"> | ||
<p style="margin-bottom: 1em;"> | <p style="margin-bottom: 1em;"> | ||
− | <i>"Always plan ahead. It wasn’t raining when Noah build the ark."</i | + | <br> |
+ | <i>"Always plan ahead. It wasn’t raining when Noah build the ark."</i> - <b>Richard Cushing</b> | ||
</p> | </p> | ||
+ | <br> | ||
<p> | <p> | ||
− | What does expanding the | + | What does expanding the Golden Gate based <a href="https://2018.igem.org/Team:Marburg/Parts" |
− | establishing the CRISPR/ | + | target="_blank">Marburg Collection</a>, automating time consuming lab work and |
+ | establishing the CRISPR/Cas12a system in <i>Synechococcus elongatus</i> UTEX 2973 have in common?<br> | ||
To achieve these objectives, it is always necessary to have a comprehensive theoretical preparation. It all | To achieve these objectives, it is always necessary to have a comprehensive theoretical preparation. It all | ||
starts with literature research, summarizing the current state of the art and based on this developing own | starts with literature research, summarizing the current state of the art and based on this developing own | ||
− | ideas. To have the theoretical background settled before the lab work starts is a key point of every project and | + | ideas. To have the theoretical background settled, before the lab work starts, is a key point of every project |
+ | and | ||
consumes many hours.<br> | consumes many hours.<br> | ||
− | Because in the near future phototrophic organisms will | + | Because in the near future phototrophic organisms will be more and more relevant for biotechnological |
applications, we want to establish the use of <i>Synechococcus elongatus</i> as a phototrophic organism for | applications, we want to establish the use of <i>Synechococcus elongatus</i> as a phototrophic organism for | ||
− | + | Synthetic Biology. Following the principles of Synthetic Biology to simplify the process of engineering of | |
biological systems, we set it our goal to establish <i>Synechococcus elongatus</i> UTEX 2973 as the fastest and | biological systems, we set it our goal to establish <i>Synechococcus elongatus</i> UTEX 2973 as the fastest and | ||
most accessible phototrophic chassis to date, providing it as a wind tunnel for phototrophic organisms with user | most accessible phototrophic chassis to date, providing it as a wind tunnel for phototrophic organisms with user | ||
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<section class="section grid"> | <section class="section grid"> | ||
<div class="sub" | <div class="sub" | ||
− | onclick="popup(' | + | onclick="popup('strain_engineering')"> |
<div class="sub-header"> | <div class="sub-header"> | ||
<h1> | <h1> | ||
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</div> | </div> | ||
<div class="sub-content"> | <div class="sub-content"> | ||
− | < | + | <div> |
− | We modified <i>Synechococcus elongatus</i> UTEX 2973 to establish the CRISPR/ | + | We modified <i>Synechococcus elongatus</i> UTEX 2973 to establish the CRISPR/Cas12a |
system in our organism. | system in our organism. | ||
− | </ | + | </div> |
</div> | </div> | ||
</div> | </div> | ||
<div class="sub" | <div class="sub" | ||
− | onclick="popup(' | + | onclick="popup('toolbox')"> |
<div class="sub-header"> | <div class="sub-header"> | ||
<h1> | <h1> | ||
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</div> | </div> | ||
<div class="sub-content"> | <div class="sub-content"> | ||
− | < | + | <div> |
We expanded last years Marburg Collection and made the parts suitable for <i>Synechococcus elongatus</i> | We expanded last years Marburg Collection and made the parts suitable for <i>Synechococcus elongatus</i> | ||
UTEX 2973. | UTEX 2973. | ||
− | </ | + | </div> |
</div> | </div> | ||
</div> | </div> | ||
− | <div id=" | + | <div id="strain_engineering" |
class="popup"> | class="popup"> | ||
<div class="popup-container"> | <div class="popup-container"> | ||
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</h1> | </h1> | ||
<button type="button" | <button type="button" | ||
− | onclick="hide(' | + | onclick="hide('strain_engineering')">X</button> |
</div> | </div> | ||
<div class="popup-content" | <div class="popup-content" | ||
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type="checkbox"> | type="checkbox"> | ||
<label for="collapsible1" | <label for="collapsible1" | ||
− | class="lbl-toggle">Natural Competence</label> | + | class="lbl-toggle"> |
+ | Natural Competence | ||
+ | <hr style="width: unset;"> | ||
+ | </label> | ||
<div class="collapsible-content"> | <div class="collapsible-content"> | ||
<div class="content-inner"> | <div class="content-inner"> | ||
<div> | <div> | ||
<p> | <p> | ||
− | As mentioned in our <a href="https://2019.igem.org/Team:Marburg/Description">description</a>, | + | As mentioned in our <a href="https://2019.igem.org/Team:Marburg/Description" |
+ | target="_blank">description</a>, | ||
<i>Synechococcus elongatus </i>UTEX 2973 is no longer naturally competent, presumably due to a | <i>Synechococcus elongatus </i>UTEX 2973 is no longer naturally competent, presumably due to a | ||
point mutation in the pilN gene ( | point mutation in the pilN gene ( | ||
− | <a href="https://www.sciencedirect.com/science/article/pii/S1096717618301757?via%3Dihub"> Li | + | <a href="https://www.sciencedirect.com/science/article/pii/S1096717618301757?via%3Dihub">Li |
− | al., 2018</a>), which means that when genetically engineering this organism other ways to | + | <i>et |
+ | al.</i>, 2018</a>), which means that when genetically engineering this organism other ways | ||
+ | to | ||
introduce exogenous DNA have to be taken into consideration. This is mainly done through | introduce exogenous DNA have to be taken into consideration. This is mainly done through | ||
electroporation or conjugation - especially triparental conjugation | electroporation or conjugation - especially triparental conjugation | ||
− | <a href="https://www.nature.com/articles/srep08132">(Yu et al., 2015)</a>. Triparental | + | <a href="https://www.nature.com/articles/srep08132">(Yu <i>et al.</i>, 2015)</a>. Triparental |
conjugation into the UTEX 2973 strain is typically performed with two <i>E. coli</i> HB101 | conjugation into the UTEX 2973 strain is typically performed with two <i>E. coli</i> HB101 | ||
− | strains, one harboring the pRL443 and one harboring the pRL623 plasmid. The latter strain is | + | strains, one harboring the pRL443 plasmid and one harboring the pRL623 plasmid. The latter |
+ | strain is | ||
then again transformed with the plasmid of interest, the prior is used as the conjugal strain - | then again transformed with the plasmid of interest, the prior is used as the conjugal strain - | ||
both have to be incubated together with the cyanobacteria for the conjugation to take place | both have to be incubated together with the cyanobacteria for the conjugation to take place | ||
<a href="https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0514-7">( | <a href="https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0514-7">( | ||
− | Wendt et al., 2016)</a>.<br> | + | Wendt <i>et al.</i>, 2016)</a>.<br> |
To overcome this time-consuming process, we planned to reintroduce natural competence into our | To overcome this time-consuming process, we planned to reintroduce natural competence into our | ||
− | strain. It was already shown | + | strain. It was already shown that this can be done by integrating an intact copy of the |
<i>pilN</i> gene into one of the neutral sites | <i>pilN</i> gene into one of the neutral sites | ||
− | <a href="https://www.sciencedirect.com/science/article/pii/S1096717618301757?via%3Dihub"> (Li | + | <a href="https://www.sciencedirect.com/science/article/pii/S1096717618301757?via%3Dihub"> (Li |
− | al., 2018)</a>, though this technique is not ideal: you have to add an antibiotic cassette in | + | <i>et |
− | order to keep selective pressure on the bacteria, so that they integrate the new gene into | + | al.</i>, 2018)</a>, though this technique is not ideal: you have to add an antibiotic |
− | + | cassette in | |
+ | order to keep selective pressure on the bacteria, so that they integrate the new gene into every | ||
+ | chromosome copy. This antibiotic resistance will persist in the strain, meaning that when | ||
further engineering the organism later on, this one resistance can not be used e.g. in vectors | further engineering the organism later on, this one resistance can not be used e.g. in vectors | ||
for transient expression - a huge downside. Furthermore, one of the neutral sites has to be | for transient expression - a huge downside. Furthermore, one of the neutral sites has to be | ||
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alt="NS bild"> | alt="NS bild"> | ||
<figcaption> | <figcaption> | ||
− | Fig.1: Approach for reintroducing the natural competence of Li et al. The pilN-Gene gets | + | Fig.1: Approach for reintroducing the natural competence of Li <i>et al.</i>, 2018. The |
− | integrated via homologous recombination into the neutral | + | pilN-Gene gets |
− | Chloramphenicol-resistance-cassette. | + | integrated via homologous recombination into the neutral site II (NS II), together with a |
+ | Chloramphenicol-resistance-cassette (cm Res). | ||
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
<p style="margin-top: 1em;"> | <p style="margin-top: 1em;"> | ||
− | Although we did not prefer this method, we still tried | + | Although we did not prefer this method, we still tried, as we were not sure, if our other |
approach would prove to be successful. We also used extensive bioinformatic tools to identify | approach would prove to be successful. We also used extensive bioinformatic tools to identify | ||
− | <a href="https://2019.igem.org/Team:Marburg/ | + | <a href="https://2019.igem.org/Team:Marburg/Model#anso" |
+ | target="_blank">new integration sites</a> in UTEX 2973, which | ||
can be used if one were to reintroduce natural competence in the above mentioned way. | can be used if one were to reintroduce natural competence in the above mentioned way. | ||
Additionally, we came up with a plan to revert the point mutation in the <i>pilN</i> gene with a | Additionally, we came up with a plan to revert the point mutation in the <i>pilN</i> gene with a | ||
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<figcaption> | <figcaption> | ||
Fig.2: Approach for reintroducing the natural competence via repairing the natural pilN-gene | Fig.2: Approach for reintroducing the natural competence via repairing the natural pilN-gene | ||
− | with Cas12a | + | with Cas12a. |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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This approach is promising, as the integration of the new <i>pilN</i> copy only enabled a low | This approach is promising, as the integration of the new <i>pilN</i> copy only enabled a low | ||
efficiency of natural transformation, which might be due to the point mutation negatively | efficiency of natural transformation, which might be due to the point mutation negatively | ||
− | affecting expression of the <i> | + | affecting expression of the <i>pilO</i> and <i>pilQ</i> genes laying downstream of <i>pilN</i> |
− | <a href="https://academic.oup.com/femsle/article/129/1/83/442013">(Li et al., 2018 ; Barten and | + | <a href="https://academic.oup.com/femsle/article/129/1/83/442013">(Li <i>et al.</i>, 2018 ; |
+ | Barten and | ||
Lill, 1995)</a>. As CRISPR/Cas12a allows accurate targeting of genetic sequences, we designed | Lill, 1995)</a>. As CRISPR/Cas12a allows accurate targeting of genetic sequences, we designed | ||
a | a | ||
− | + | gRNA leading the Cas12a protein to the <i>pilN</i> locus. The repair template was taken from | |
− | the <i>S. elongatus</i> PCC | + | the <i>S. elongatus</i> PCC 7942 genome, where the gene is still intact, allowing the cell to |
repair the cut introduced by Cas12a accordingly, reversing the point mutation, which leads to an | repair the cut introduced by Cas12a accordingly, reversing the point mutation, which leads to an | ||
intact copy of <i>pilN</i> again - a more elegant approach than simply inserting a second copy | intact copy of <i>pilN</i> again - a more elegant approach than simply inserting a second copy | ||
− | of the gene. As our own CRISPR system was still | + | of the gene. As our own CRISPR system was still under construction at that point, we had to rely |
+ | on | ||
pSL2680, a replicating base vector for constructing CRISPR/Cas12a editing plasmids by | pSL2680, a replicating base vector for constructing CRISPR/Cas12a editing plasmids by | ||
<a href="https://www.nature.com/articles/srep39681">Ungerer and Pakrasi, 2016</a>.<br> | <a href="https://www.nature.com/articles/srep39681">Ungerer and Pakrasi, 2016</a>.<br> | ||
We followed their protocol <a href="https://www.addgene.org/85581">(available here on | We followed their protocol <a href="https://www.addgene.org/85581">(available here on | ||
− | Addgene)</a>, annealing oligos to construct the | + | Addgene)</a>, annealing oligos to construct the gRNA. Small overhangs were added to enable |
the | the | ||
ligation into the AarI-digested vector, where a <i>lacZ</i> cassette was replaced, which allowed | ligation into the AarI-digested vector, where a <i>lacZ</i> cassette was replaced, which allowed | ||
− | for blue/white screening of recombinant colonies | + | for blue/white screening of recombinant colonies. Additionally, the repair template had to be |
− | + | constructed by PCR with added overhangs for | |
the following Gibson reaction. As stated, it was taken from the <i>S. elongatus</i> PCC 7942 | the following Gibson reaction. As stated, it was taken from the <i>S. elongatus</i> PCC 7942 | ||
genome. It was designed in such a way that the point mutation inside the UTEX 2973 genome was | genome. It was designed in such a way that the point mutation inside the UTEX 2973 genome was | ||
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alt="blub"> | alt="blub"> | ||
<figcaption> | <figcaption> | ||
− | Fig.3: The | + | Fig.3: The top of the figure shows a part of the pilN gene of UTEX 2973. You can |
− | clearly see the | + | clearly see the mutation which causes the STOP-Codon. Additionally the PAM-sequence and the |
− | target region of Cas12a are visible. The sequence in the middle is from the PCC 7942. This | + | target region of Cas12a are visible. The sequence in the middle is from the strain PCC 7942. |
+ | This | ||
sequence differs in just one basepair, but this basepair has the effect, that the pilN gene is | sequence differs in just one basepair, but this basepair has the effect, that the pilN gene is | ||
working in PCC 7942. Additionally the PAM-site of Cas12a does not appear in PCC 7942, due to | working in PCC 7942. Additionally the PAM-site of Cas12a does not appear in PCC 7942, due to | ||
Line 255: | Line 201: | ||
</figure> | </figure> | ||
<p style="margin-top: 1em;"> | <p style="margin-top: 1em;"> | ||
− | After the successful Gibson assembly of | + | After the successful Gibson assembly of gRNA and repair template into the Cas12a carrying |
vector, nearly two weeks had passed, indicating that working with this vector can be quite | vector, nearly two weeks had passed, indicating that working with this vector can be quite | ||
tedious and time consuming. This is one of the many reasons why we chose to implement such a | tedious and time consuming. This is one of the many reasons why we chose to implement such a | ||
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type="checkbox"> | type="checkbox"> | ||
<label for="collapsible2" | <label for="collapsible2" | ||
− | class="lbl-toggle">CRISPR gene editing</label> | + | class="lbl-toggle"> |
+ | CRISPR gene editing | ||
+ | <hr style="width: unset;"> | ||
+ | </label> | ||
<div class="collapsible-content"> | <div class="collapsible-content"> | ||
<div class="content-inner"> | <div class="content-inner"> | ||
<p> | <p> | ||
− | CRISPR/Cas systems are powerful tools that have gained a lot of popularity in | + | CRISPR/Cas systems are powerful tools that have gained a lot of popularity in recent years. |
As they can be used for a wide array of applications - like the integration of whole genes, | As they can be used for a wide array of applications - like the integration of whole genes, | ||
− | + | alternation of single nucleotides, knock-outs of whole genetic regions, as well as the use of the | |
DNA-binding property in a multitude of applications through so called deadCas systems, where the | DNA-binding property in a multitude of applications through so called deadCas systems, where the | ||
Cas protein does not exhibit nuclease activity | Cas protein does not exhibit nuclease activity | ||
− | <a href="https://www.cell.com/action/showPdf?pii=S0092-8674%2814%2900604-7">( | + | <a href="https://www.cell.com/action/showPdf?pii=S0092-8674%2814%2900604-7">(Hsu <i>et al.</i>, |
+ | 2014)</a> | ||
- we were eager to implement such a system into our own | - we were eager to implement such a system into our own | ||
− | <a href="https://2019.igem.org/Team:Marburg/Design">toolbox</a>. Diving into | + | <a href="https://2019.igem.org/Team:Marburg/Design#toolbox">toolbox</a>. Diving into literature we |
− | noticed many different systems are available,the most commonly used one being CRISPR/Cas9, and we | + | noticed many different systems are available, the most commonly used one being CRISPR/Cas9, and we |
began to wonder which of them we should use.<br> | began to wonder which of them we should use.<br> | ||
− | In our <a href="https://2019.igem.org/Team:Marburg/Description">description</a> we presented | + | In our <a href="https://2019.igem.org/Team:Marburg/Description#strain_engineering">description</a> |
+ | we presented | ||
CRISPR/Cas9 and CRISPR/Cas12a, showing the differences of these two systems. Looking deeper into | CRISPR/Cas9 and CRISPR/Cas12a, showing the differences of these two systems. Looking deeper into | ||
CRISPR/Cas12a we noticed a few advantages that finally led us to choose it as our preferred | CRISPR/Cas12a we noticed a few advantages that finally led us to choose it as our preferred | ||
Line 296: | Line 247: | ||
features of these CRISPR/Cas systems, but how to actually apply it differs:<br> | features of these CRISPR/Cas systems, but how to actually apply it differs:<br> | ||
For Cas9 each sgRNA is in need of its own promoter, which means that they have to be expressed | For Cas9 each sgRNA is in need of its own promoter, which means that they have to be expressed | ||
− | from different vectors or a multi cassette vector ( | + | from different vectors or a multi cassette vector (<a |
− | + | href="https://onlinelibrary.wiley.com/doi/full/10.1002/wrna.1481#wrna1481-bib-0058">Swarts & | |
− | + | Jinek, 2018</a>; | |
<a href="https://onlinelibrary.wiley.com/doi/full/10.1002/wrna.1481#wrna1481-bib-0104"> Z. Zhang | <a href="https://onlinelibrary.wiley.com/doi/full/10.1002/wrna.1481#wrna1481-bib-0104"> Z. Zhang | ||
− | et al., 2016 </a> ). In contrary, multiplexed genome editing with Cas12a can be achieved simply | + | <i>et al.</i>, 2016</a>). In contrary, multiplexed genome editing with Cas12a can be achieved |
− | by expressing all of the needed guide RNAs in one transcriptional unit, | + | simply |
− | + | by expressing all of the needed guide RNAs in one transcriptional unit, the crRNA. | |
− | + | This is a huge advantage of Cas12a. Furthermore, | |
− | + | ||
CRISPR/Cas9 was shown to be toxic in cyanobacteria | CRISPR/Cas9 was shown to be toxic in cyanobacteria | ||
<a href="https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0514-7"> | <a href="https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0514-7"> | ||
− | (Wendt et al., 2016)</a>, which is one of the foremost reasons CRISPR technologies have not been | + | (Wendt <i>et al.</i>, 2016)</a>, which is one of the foremost reasons CRISPR technologies have |
− | widely applied in cyanobacteria - the usage of Cas12a though, | + | not been |
− | + | widely applied in cyanobacteria - the usage of Cas12a though, seems to be less | |
+ | toxic <a href="https://www.nature.com/articles/srep39681"> (Ungerer and Pakrasi, 2016)</a>, | ||
making it the ideal candidate for the Green Extension of the | making it the ideal candidate for the Green Extension of the | ||
<a href="https://2019.igem.org/Team:Marburg/Parts">Marburg Collection</a>.<br> | <a href="https://2019.igem.org/Team:Marburg/Parts">Marburg Collection</a>.<br> | ||
The actual implementation of the CRISPR/Cas12a system into our toolbox necessitated a well thought | The actual implementation of the CRISPR/Cas12a system into our toolbox necessitated a well thought | ||
out plan. The design of our CRISPR/Cas12a system was mainly affected by the fact that we wanted to | out plan. The design of our CRISPR/Cas12a system was mainly affected by the fact that we wanted to | ||
− | have a convenient and rapid tool for genomic manipulation. The lvl 0 | + | have a convenient and rapid tool for genomic manipulation. The lvl 0 <a |
− | + | href="https://2019.igem.org/Team:Marburg/Basic_Part">part</a> of the | |
Cas12a protein was created via PCR amplification from the plasmid pSL2680, but special overhangs | Cas12a protein was created via PCR amplification from the plasmid pSL2680, but special overhangs | ||
were added in order to clone the PCR product into a lvl 0 acceptor vector. The part was introduced | were added in order to clone the PCR product into a lvl 0 acceptor vector. The part was introduced | ||
Line 323: | Line 274: | ||
used for lvl 1 assembly were: pMC0_1_03 + pMC0_2_03 + pMC0_3_07 + pMC0_4_33 + pMC0_5_07 + | used for lvl 1 assembly were: pMC0_1_03 + pMC0_2_03 + pMC0_3_07 + pMC0_4_33 + pMC0_5_07 + | ||
pMC0_6_17. For the construction of the crRNA part the design of the plasmid pSL2680 was mainly | pMC0_6_17. For the construction of the crRNA part the design of the plasmid pSL2680 was mainly | ||
− | maintained, but the <i>lacZ</i> cassette was replaced by a GFP cassette to enable easier screening | + | maintained, but the <i>lacZ</i> cassette was replaced by a GFP cassette with BsmBI cutting sites |
− | of | + | to enable easier screening |
+ | of gRNA assembly and to reduce expenses for X-Gal/IPTG. It was constructed as a part reaching | ||
from the RBS site to the end of the terminator site. As the whole system is built for modular | from the RBS site to the end of the terminator site. As the whole system is built for modular | ||
cloning in PhytoBrick syntax, it is possible to freely exchange the parts around the Cas12a and | cloning in PhytoBrick syntax, it is possible to freely exchange the parts around the Cas12a and | ||
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type="checkbox"> | type="checkbox"> | ||
<label for="collapsible3" | <label for="collapsible3" | ||
− | class="lbl-toggle">Cyanobacterial shuttle vectors</label> | + | class="lbl-toggle"> |
+ | Cyanobacterial shuttle vectors | ||
+ | <hr style="width: unset;"> | ||
+ | </label> | ||
<div class="collapsible-content"> | <div class="collapsible-content"> | ||
<div class="content-inner"> | <div class="content-inner"> | ||
<div> | <div> | ||
− | + | <div style="display: flex; flex-direction: row;"> | |
− | + | ||
+ | <p> | ||
+ | Following our dream to create the most versatile, MoClo compatible shuttle vector for | ||
cyanobacteria we made sure to pay attention to detail. When creating new shuttle vectors, one of | cyanobacteria we made sure to pay attention to detail. When creating new shuttle vectors, one of | ||
the most important points to consider is the replication element that is being used, mainly due | the most important points to consider is the replication element that is being used, mainly due | ||
to the wanted copy number and a phenomenon called plasmid incompatibility. Plasmids harboring | to the wanted copy number and a phenomenon called plasmid incompatibility. Plasmids harboring | ||
the same replication or partitioning system can often not be stably maintained in a cell - they | the same replication or partitioning system can often not be stably maintained in a cell - they | ||
− | are incompatible <a href="https://doi.org/10.1016/0147-619X(78)90001-X">(Novick | + | are incompatible <a href="https://doi.org/10.1016/0147-619X(78)90001-X">(Novick & |
Hoppenstaedt, 1978)</a>. With multiple different plasmids bearing the same replication | Hoppenstaedt, 1978)</a>. With multiple different plasmids bearing the same replication | ||
elements, | elements, | ||
the replication machinery will randomly choose which plasmids to replicate, leading to one of | the replication machinery will randomly choose which plasmids to replicate, leading to one of | ||
the different plasmids being copied more frequently than the other | the different plasmids being copied more frequently than the other | ||
− | + | . As we used the minimal replication element from the pANS plasmid | |
of <i>S. elongatus</i> in our shuttle-vectors, in order to have a native origin of replication, | of <i>S. elongatus</i> in our shuttle-vectors, in order to have a native origin of replication, | ||
we had to consider such plasmid incompatibilities and made sure to | we had to consider such plasmid incompatibilities and made sure to | ||
− | <a href="https://2019.igem.org/Team:Marburg/Results">cure our strain</a> of the endogenous pANS | + | <a href="https://2019.igem.org/Team:Marburg/Results#strain_engineering">cure our strain</a> of |
− | - which we could successfully | + | the endogenous pANS |
+ | - which we could successfully proove.<br> </p> | ||
+ | |||
+ | |||
+ | |||
+ | <figure style="text-align: center; margin-left: 25px; min-width:400px"> | ||
+ | |||
+ | <img | ||
+ | src="https://static.igem.org/mediawiki/2019/9/91/T--Marburg--Plasmid_curing.svg" | ||
+ | alt="Figure 4 -Schematic procedure of curing the natural plasmid pANS"> | ||
+ | <figcaption style="max-width: 500px; text-align: center"> | ||
+ | Figure 4 -Schematic procedure of curing the natural plasmid pANS | ||
+ | </figcaption> </figure> | ||
+ | |||
+ | <br> | ||
+ | </div> | ||
+ | <p> | ||
+ | |||
The next step was the creation of our own, modular shuttle vector. For this we had to pay | The next step was the creation of our own, modular shuttle vector. For this we had to pay | ||
attention, as we had to fit it to the PhytoBrick standard in order to use it in our Green | attention, as we had to fit it to the PhytoBrick standard in order to use it in our Green | ||
Line 381: | Line 355: | ||
introduced. Due to the length and complexity of the sequence we had to divide the synthesis of | introduced. Due to the length and complexity of the sequence we had to divide the synthesis of | ||
the minimal replication element into three parts that later had to be fused together. | the minimal replication element into three parts that later had to be fused together. | ||
− | Additionally, we wanted to implement a reporter for easy selection. We chose | + | Additionally, we wanted to implement a reporter for easy selection. We chose RFP, which was |
− | amplified out of the Lvl0_8_Amp/ColE1 part from last years Marburg Collection in addition with | + | amplified out of the <a href="https://2018.igem.org/Team:Marburg/Parts target=" |
+ | _blank">Lvl0_8_Amp/ColE1</a> part from last years Marburg Collection in addition with | ||
the ColE1 ori that can be found on it. This means that our vector does not just contain the | the ColE1 ori that can be found on it. This means that our vector does not just contain the | ||
cyanobacterial ori of our strain, but also a high copy origin for replication in <i>E. coli</i> | cyanobacterial ori of our strain, but also a high copy origin for replication in <i>E. coli</i> | ||
− | <a href="https://doi.org/10.1016/S0065-2660(02)46013-0">(Gerhart et al., 2002)</a>. As an | + | <a href="https://doi.org/10.1016/S0065-2660(02)46013-0">(Gerhart <i>et al.</i>, 2002)</a>. As an |
antibiotic cassette we chose spectinomycin, which we also amplified by PCR, this time from | antibiotic cassette we chose spectinomycin, which we also amplified by PCR, this time from | ||
pAM4787 | pAM4787 | ||
<a href="https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000377">(Chen | <a href="https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.000377">(Chen | ||
− | et al., 2016)</a>. Finally, those five fragments - the three parts of the minimal replication | + | <i>et al.</i>, 2016)</a>. Finally, those five fragments - the three parts of the minimal |
− | element, the ColE1 ori & | + | replication |
+ | element, the ColE1 ori & RFP cassette and the spectinomycin resistance cassette (aadA) - were | ||
fused together in a Gibson reaction, resulting in BBa_K3228069 (sometimes also called lvl 1 | fused together in a Gibson reaction, resulting in BBa_K3228069 (sometimes also called lvl 1 | ||
ori), the first cyanobacterial shuttle vector for cloning lvl 1 constructs in a modular way. | ori), the first cyanobacterial shuttle vector for cloning lvl 1 constructs in a modular way. | ||
− | This part has two BsaI sites that flank the | + | This part has two BsaI sites that flank the RFP cassette, so that this genetic element will be |
exchanged with the other parts in a lvl 1 Golden Gate reaction. | exchanged with the other parts in a lvl 1 Golden Gate reaction. | ||
</p> | </p> | ||
Line 406: | Line 382: | ||
<p style="margin-top: 1em;"> | <p style="margin-top: 1em;"> | ||
In addition to this, we created a second shuttle vector, this time for cloning lvl 2 constructs: | In addition to this, we created a second shuttle vector, this time for cloning lvl 2 constructs: | ||
− | This vector has mostly the same design as BBa_K3228069, but the | + | This vector has mostly the same design as BBa_K3228069, but the RFP cassette is flanked by BsmBI |
sites, enabling the construction of lvl 2 vectors. | sites, enabling the construction of lvl 2 vectors. | ||
</p> | </p> | ||
Line 412: | Line 388: | ||
<img style="height: 50ex; width: 50ex;" | <img style="height: 50ex; width: 50ex;" | ||
src="https://static.igem.org/mediawiki/2019/3/37/T--Marburg--Toolbox_Shuttle_Lvl2.svg" | src="https://static.igem.org/mediawiki/2019/3/37/T--Marburg--Toolbox_Shuttle_Lvl2.svg" | ||
− | alt=" | + | alt="lv2"> |
<figcaption> | <figcaption> | ||
− | Fig. 5: Schematic picture of the cyanobacterial shuttle vector for cloning lvl | + | Fig. 5: Schematic picture of the cyanobacterial shuttle vector for cloning lvl 2 constructs. |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
Line 420: | Line 396: | ||
Furthermore this part bears a kanamycin resistance cassette instead of the spectinomycin | Furthermore this part bears a kanamycin resistance cassette instead of the spectinomycin | ||
resistance of the lvl 1 ori. This part was assembled in a four part Gibson reaction, as in | resistance of the lvl 1 ori. This part was assembled in a four part Gibson reaction, as in | ||
− | addition to ColE1 and the | + | addition to ColE1 and the RFP cassette also the kanamycin resistance cassette could be amplified |
via PCR, in this case from pYTK_0_84, a plasmid from the Dueber MoClo Yeast Toolkit, resulting | via PCR, in this case from pYTK_0_84, a plasmid from the Dueber MoClo Yeast Toolkit, resulting | ||
in BBa_K3228089 (sometimes called lvl 2 ori). For all these cloning processes special overhangs | in BBa_K3228089 (sometimes called lvl 2 ori). For all these cloning processes special overhangs | ||
Line 433: | Line 409: | ||
</div> | </div> | ||
</div> | </div> | ||
− | <div id=" | + | <div id="toolbox" |
class="popup"> | class="popup"> | ||
<div class="popup-container"> | <div class="popup-container"> | ||
Line 441: | Line 417: | ||
</h1> | </h1> | ||
<button type="button" | <button type="button" | ||
− | onclick="hide(' | + | onclick="hide('toolbox')">X</button> |
</div> | </div> | ||
<div class="popup-content" | <div class="popup-content" | ||
Line 451: | Line 427: | ||
type="checkbox"> | type="checkbox"> | ||
<label for="collapsiblebal1" | <label for="collapsiblebal1" | ||
− | class="lbl-toggle">The Marburg Collection: | + | class="lbl-toggle"> |
+ | The Marburg Collection: A recap | ||
+ | <hr style="width: unset;"> | ||
+ | </label> | ||
<div class="collapsible-content"> | <div class="collapsible-content"> | ||
<div class="content-inner"> | <div class="content-inner"> | ||
<p> | <p> | ||
− | The Marburg Collection is a toolbox from last year’s iGEM Marburg team for the rational design of | + | The Marburg Collection is a toolbox from <a href="https://2018.igem.org/Team:Marburg" |
+ | target="_blank">last year’s iGEM Marburg team</a> for the rational design of | ||
metabolic pathways and genetic circuits or any other DNA construct. Thanks to its flexible design | metabolic pathways and genetic circuits or any other DNA construct. Thanks to its flexible design | ||
based on the ‘Dueber toolbox’ design from <a href="https://www.ncbi.nlm.nih.gov/pubmed/25871405"> | based on the ‘Dueber toolbox’ design from <a href="https://www.ncbi.nlm.nih.gov/pubmed/25871405"> | ||
− | Lee et | + | Lee <i>et al.</i> (2015)</a> it can be used in a multitude of chassis: since it complies with |
+ | the | ||
PhytoBrick standard, it can even be extended to eukaryotic chassis such as plants. The design of | PhytoBrick standard, it can even be extended to eukaryotic chassis such as plants. The design of | ||
− | the toolbox is rather simple and user friendly: | + | the toolbox is rather simple and user friendly: lvl 0 parts are the basic foundation of every |
− | assembly. They contain a single genetic element such as a promoter or terminator. Up to 8 | + | assembly. They contain a single genetic element such as a promoter or terminator. Up to 8 lvl 0 |
− | parts are used to build a | + | parts are used to build a lvl1 plasmid containing a single transcription unit. Up to 5 of these |
− | transcription units can be assembled together in a | + | transcription units can be assembled together in a lvl 2 plasmid |
− | <a href="https://www.ncbi.nlm.nih.gov/pubmed/25871405">(Lee et | + | <a href="https://www.ncbi.nlm.nih.gov/pubmed/25871405">(Lee <i>et al.</i>, 2015)</a>. |
</p> | </p> | ||
<figure style="text-align: center;"> | <figure style="text-align: center;"> | ||
Line 471: | Line 452: | ||
alt="Level 1-Level 2-assembly"> | alt="Level 1-Level 2-assembly"> | ||
<figcaption style="max-width: 2400px; text-align: center;"> | <figcaption style="max-width: 2400px; text-align: center;"> | ||
− | Fig.1 - Level 1 | + | Fig.1 - Level 1 & Level 2 -assembly |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
Line 482: | Line 463: | ||
type="checkbox"> | type="checkbox"> | ||
<label for="collapsiblebla2" | <label for="collapsiblebla2" | ||
− | class="lbl-toggle">Design of placeholders</label> | + | class="lbl-toggle"> |
+ | Design of placeholders | ||
+ | <hr style="width: unset;"> | ||
+ | </label> | ||
<div class="collapsible-content"> | <div class="collapsible-content"> | ||
<div class="content-inner"> | <div class="content-inner"> | ||
<p> | <p> | ||
− | Here we present a new feature of the Marburg Collection 2.0: | + | Here we present a new feature of the Marburg Collection 2.0: placeholders. These parts make it |
possible to construct plasmids with a placeholder, which can be later on exchanged with any part | possible to construct plasmids with a placeholder, which can be later on exchanged with any part | ||
of the same type.<br> | of the same type.<br> | ||
A key feature in our expansion is the addition of placeholders that allow high throughput assembly | A key feature in our expansion is the addition of placeholders that allow high throughput assembly | ||
− | of plasmids that only differ in one part. A promoter placeholder for example is built into a | + | of plasmids that only differ in one part. A promoter placeholder for example is built into a lvl 1 |
construct at the promoter position. Instead of a promoter however it contains a GFP cassette and | construct at the promoter position. Instead of a promoter however it contains a GFP cassette and | ||
reversed BsaI cutting sites. This allows BsaI cleavage and removal of the GFP cassette even after | reversed BsaI cutting sites. This allows BsaI cleavage and removal of the GFP cassette even after | ||
Line 496: | Line 480: | ||
</p> | </p> | ||
<p style="margin-top: 1em;"> | <p style="margin-top: 1em;"> | ||
− | After that any promoter of choice can be inserted at that position. After ligation, no BsaI | + | After that, any promoter of choice can be inserted at that position. After ligation, no BsaI |
− | cutting sites remain on the vector, so in the end mainly the newly assembled | + | cutting sites remain on the vector, so in the end mainly the newly assembled remain. These steps |
also happen in a one pot one step reaction just like any other Golden Gate assembly. | also happen in a one pot one step reaction just like any other Golden Gate assembly. | ||
</p> | </p> | ||
Line 509: | Line 493: | ||
</figure> | </figure> | ||
<p style="margin-top: 1em;"> | <p style="margin-top: 1em;"> | ||
− | White green selection under UV light can be used to determine the colonies with the right plasmid: | + | White/green selection under UV light can be used to determine the colonies with the right plasmid: |
green ones still contain the plasmid with a placeholder, white ones contain the desired vector. | green ones still contain the plasmid with a placeholder, white ones contain the desired vector. | ||
</p> | </p> | ||
Line 520: | Line 504: | ||
type="checkbox"> | type="checkbox"> | ||
<label for="collapsible2na" | <label for="collapsible2na" | ||
− | class="lbl-toggle">Available Placeholders</label> | + | class="lbl-toggle"> |
+ | Available Placeholders | ||
+ | <hr style="width: unset;"> | ||
+ | </label> | ||
<div class="collapsible-content"> | <div class="collapsible-content"> | ||
<div class="content-inner"> | <div class="content-inner"> | ||
Line 529: | Line 516: | ||
for a specific application. This however would then be a three part assembly. The application of | for a specific application. This however would then be a three part assembly. The application of | ||
such parts is so narrow that we decided to build the most useful ones. Thanks to its clever | such parts is so narrow that we decided to build the most useful ones. Thanks to its clever | ||
− | design the construction of more placeholder is so simple that it can be done by the user | + | design the construction of more placeholder is so simple that it can be done by the user itself |
with a single site directed mutagenesis of a flank. | with a single site directed mutagenesis of a flank. | ||
</p> | </p> | ||
Line 542: | Line 529: | ||
class="lbl-toggle"> | class="lbl-toggle"> | ||
Design of the first panS based MoClo compatible shuttle vector | Design of the first panS based MoClo compatible shuttle vector | ||
+ | <hr style="width: unset;"> | ||
</label> | </label> | ||
<div class="collapsible-content"> | <div class="collapsible-content"> | ||
<div class="content-inner"> | <div class="content-inner"> | ||
<p> | <p> | ||
− | The heart piece of | + | The heart piece of the Green Expansion is <a href="http://parts.igem.org/Part:BBa_K3228069" |
+ | target="_blank">BBa_K3228069</a>, a lvl 0 part containing origins of replication | ||
for <i>E. coli</i> and <i>S. elongatus</i> as well as a spectinomycin cassette. It resembles a | for <i>E. coli</i> and <i>S. elongatus</i> as well as a spectinomycin cassette. It resembles a | ||
type 7+8 (antibiotic cassette + ori) composite part and can be seen as the cyanobacteria specific | type 7+8 (antibiotic cassette + ori) composite part and can be seen as the cyanobacteria specific | ||
− | + | lvl1 entry vector. Another version of this entry vector contains a kanamycin cassette and BsmbI | |
− | cutting sites and can be used as the | + | cutting sites and can be used as the lvl2 entry vector. Just like in our lvl0 entry vectors for |
basic parts, we prompted for a fluorescence based reporter in the dropout, rather than lacZ for | basic parts, we prompted for a fluorescence based reporter in the dropout, rather than lacZ for | ||
blue/white screening. Therefore both vectors contain an RFP dropout to signal an insertion. Using | blue/white screening. Therefore both vectors contain an RFP dropout to signal an insertion. Using | ||
Line 561: | Line 550: | ||
alt="Shuttle lvl 1"> | alt="Shuttle lvl 1"> | ||
<figcaption style="max-width: 2400px; text-align: center;"> | <figcaption style="max-width: 2400px; text-align: center;"> | ||
− | Fig.4 - Shuttle | + | Fig.4 - Shuttle lvl1 |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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type="checkbox"> | type="checkbox"> | ||
<label for="collapsibleih2" | <label for="collapsibleih2" | ||
− | class="lbl-toggle">Designing the characterization of BioBricks</label> | + | class="lbl-toggle"> |
+ | Designing the characterization of BioBricks | ||
+ | <hr style="width: unset;"> | ||
+ | </label> | ||
<div class="collapsible-content"> | <div class="collapsible-content"> | ||
<div class="content-inner"> | <div class="content-inner"> | ||
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principles of Synthetic Biology:<br>In order to be comparable, all of the constructs must be | principles of Synthetic Biology:<br>In order to be comparable, all of the constructs must be | ||
almost identical and only differ in the part to be tested. Instead of building each construct | almost identical and only differ in the part to be tested. Instead of building each construct | ||
− | independently we utilized our | + | independently we utilized our <a |
− | + | href="https://2019.igem.org/Team:Marburg/Results#marburg_collection" | |
+ | target="_blank">placeholders</a> to build all | ||
measurement plasmids for the same type of part from the same blueprint.<br>We present a set of | measurement plasmids for the same type of part from the same blueprint.<br>We present a set of | ||
measurement entry vectors for the characterization of BioBricks in cyanobacteria (Part range | measurement entry vectors for the characterization of BioBricks in cyanobacteria (Part range | ||
BBa_K3228073 to BBa_K3228075 as well as BBa_K3228090). They contain our MoClo compatible shuttle | BBa_K3228073 to BBa_K3228075 as well as BBa_K3228090). They contain our MoClo compatible shuttle | ||
− | vector for cyanobacteria BBa_K3228069 and are therefore the only MoClo based vector for the | + | vector for cyanobacteria <a href="parts.igem.org/Parts:BBa_K3228069" |
− | characterization of BioBricks in cyanobacteria. These pre assembled | + | target="_blank">BBa_K3228069</a> and are therefore the only MoClo based vector for the |
− | placeholder for their respective BioBrick type that acts as a | + | characterization of BioBricks in cyanobacteria. These pre assembled lvl1 plasmids contain a |
− | insert any part of the same type for an easy characterization. In our results we show how these | + | placeholder for their respective BioBrick type that acts as a dropout to quickly and effortlessly |
+ | insert any part of the same type for an easy characterization. In our <a | ||
+ | href="https://2019.igem.org/Team:Marburg/Results" | ||
+ | target="_blank">results</a> we show how these | ||
measurement entry vectors can save a lot of effort and money when characterizing a greater library | measurement entry vectors can save a lot of effort and money when characterizing a greater library | ||
of parts. Additionally, the usage of the same entry vector for each measurement will aid in | of parts. Additionally, the usage of the same entry vector for each measurement will aid in | ||
Line 613: | Line 609: | ||
signal. On top of that, measuring the activity both upstream and downstream of the terminator with | signal. On top of that, measuring the activity both upstream and downstream of the terminator with | ||
two independent reporters would give insight on the exact transcriptional activity around the area | two independent reporters would give insight on the exact transcriptional activity around the area | ||
− | of the terminator <a href="https://www.ncbi.nlm.nih.gov/pubmed/23868258">(Chen et al., 2013)</a>, | + | of the terminator <a href="https://www.ncbi.nlm.nih.gov/pubmed/23868258">(Chen <i>et al.</i>, |
+ | 2013)</a>, | ||
resulting in the most accurate results in respect to the molecular dynamics of a terminator | resulting in the most accurate results in respect to the molecular dynamics of a terminator | ||
− | (see <a href="https://2019.igem.org/Team:Marburg/Model">modeling</a>).<br> | + | (see <a href="https://2019.igem.org/Team:Marburg/Model#terminator_model">modeling</a>).<br> |
− | A | + | A lvl2 plasmid was logically the easiest way to construct such a part. We designed a normal lvl1 |
− | plasmid containing an mTurqouise reporter and a secondary | + | plasmid containing an mTurqouise reporter and a secondary lvl1 plasmid containing an YFP reporter |
but missing a promoter. | but missing a promoter. | ||
</p> | </p> | ||
Line 624: | Line 621: | ||
alt="Measurement vectors - Terminator"> | alt="Measurement vectors - Terminator"> | ||
<figcaption style="max-width: 2400px; text-align: center;"> | <figcaption style="max-width: 2400px; text-align: center;"> | ||
− | Fig.6 - Measurement vectors - | + | Fig.6 - Measurement vectors - terminator |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
<p style="margin-top: 1em;"> | <p style="margin-top: 1em;"> | ||
The fraction of the signal strength of YFP and mTurquoise describe the isolative capacity of the | The fraction of the signal strength of YFP and mTurquoise describe the isolative capacity of the | ||
− | terminator best <a href="https://www.ncbi.nlm.nih.gov/pubmed/23868258">(Chen et al., | + | terminator best <a href="https://www.ncbi.nlm.nih.gov/pubmed/23868258">(Chen <i>et al.</i>, |
2013)</a>.<br> | 2013)</a>.<br> | ||
This way of calculating isolative strength is also used in RNA-seq to determine the strength of | This way of calculating isolative strength is also used in RNA-seq to determine the strength of | ||
terminators. | terminators. | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
</p> | </p> | ||
</div> | </div> | ||
Line 661: | Line 641: | ||
class="lbl-toggle"> | class="lbl-toggle"> | ||
Finding new artificial Neutral integration Site options (a.N.S.o.) | Finding new artificial Neutral integration Site options (a.N.S.o.) | ||
+ | <hr style="width: unset;"> | ||
</label> | </label> | ||
<div class="collapsible-content"> | <div class="collapsible-content"> | ||
<div class="content-inner"> | <div class="content-inner"> | ||
<p> | <p> | ||
− | Artificial neutral integration | + | Artificial neutral integration site options (aNSo) for our purpose in <i>Synechococcus |
− | + | elongatus</i> needed to fulfill three criteria, to be genuinely considered as potential | |
candidates.<br> | candidates.<br> | ||
A highly precise algorithm was implemented in a Python script to find these potential candidates | A highly precise algorithm was implemented in a Python script to find these potential candidates | ||
− | (see <a href="https://2019.igem.org/Team:Marburg/Model">modeling</a>) by describing the following | + | (see <a href="https://2019.igem.org/Team:Marburg/Model#anso" |
+ | target="_blank">modeling</a>) by describing the following | ||
criteria. First, no gene and transcription start site (TSS), i.e. no CDS, was allowed to be | criteria. First, no gene and transcription start site (TSS), i.e. no CDS, was allowed to be | ||
disturbed, assuring that no lethal modification was created by integration. Thereby, we searched | disturbed, assuring that no lethal modification was created by integration. Thereby, we searched | ||
Line 681: | Line 663: | ||
compatible. All sequences that contained such restriction site were discarded. Executing this | compatible. All sequences that contained such restriction site were discarded. Executing this | ||
newly developed and unique algorithm resulted in two unique aNSo's within the genome of <i>S. | newly developed and unique algorithm resulted in two unique aNSo's within the genome of <i>S. | ||
− | + | elongatus</i>. | |
</p> | </p> | ||
<figure style="text-align: center;"> | <figure style="text-align: center;"> | ||
Line 699: | Line 681: | ||
type="checkbox"> | type="checkbox"> | ||
<label for="collapsible1002" | <label for="collapsible1002" | ||
− | class="lbl-toggle">Design neutral integration sites</label> | + | class="lbl-toggle"> |
+ | Design neutral integration sites | ||
+ | <hr style="width: unset;"> | ||
+ | </label> | ||
<div class="collapsible-content"> | <div class="collapsible-content"> | ||
<div class="content-inner"> | <div class="content-inner"> | ||
Line 719: | Line 704: | ||
<li> | <li> | ||
<b>Step 1:</b> Find your integration site.<br> | <b>Step 1:</b> Find your integration site.<br> | ||
− | For more on this see <a href="https://2019.igem.org/Team:Marburg/Model">Modeling: integration | + | For more on this see <a href="https://2019.igem.org/Team:Marburg/Model#anso">Modeling: |
− | + | integration | |
+ | sites</a> | ||
</li> | </li> | ||
<li> | <li> | ||
Line 738: | Line 724: | ||
</li> | </li> | ||
<li> | <li> | ||
− | <b>Step 5:</b> | + | <b>Step 5:</b> Digest your PCR sample with BsaI (Note that this is uncommon for LVL 0 Cloning |
but necessary because of the internal BsmBI restriction site) | but necessary because of the internal BsmBI restriction site) | ||
</li> | </li> | ||
<li> | <li> | ||
− | <b>Step 6:</b> Digest your | + | <b>Step 6:</b> Digest your lvl0 Entry vector with BsmBI and purify it over an agarose gel to |
remove the GfP Dropout. | remove the GfP Dropout. | ||
</li> | </li> | ||
<li> | <li> | ||
− | <b>Step 7:</b> Ligate your digested PCR sample and | + | <b>Step 7:</b> Ligate your digested PCR sample and lvl0 Entry vector overnight. |
</li> | </li> | ||
<li> | <li> | ||
<b>Step 8:</b> Transform your ligation as usual in an <i>E. coli</i> or <i>V. natriegens</i> | <b>Step 8:</b> Transform your ligation as usual in an <i>E. coli</i> or <i>V. natriegens</i> | ||
− | strain for cloning. Thanks to the predigested | + | strain for cloning. Thanks to the predigested lvl0 entry vector most colonies should appear |
white. Pick a few colonies and verify the construct inside by sequencing. Usually at least 1 | white. Pick a few colonies and verify the construct inside by sequencing. Usually at least 1 | ||
in 2 sequencing results yields the correct construct. | in 2 sequencing results yields the correct construct. | ||
Line 757: | Line 743: | ||
</div> | </div> | ||
<p style="margin-top: 1em;"> | <p style="margin-top: 1em;"> | ||
− | In a | + | In a lvl1 construct, the positions 2-5 representing a full transcription unit (promoter, RBS, |
CDS, terminator) would be integrated into the genome, while positions 7-8 (origin of replication, | CDS, terminator) would be integrated into the genome, while positions 7-8 (origin of replication, | ||
antibiotic cassette) would be cut off in the recombination event. The issue with this assembly | antibiotic cassette) would be cut off in the recombination event. The issue with this assembly | ||
Line 769: | Line 755: | ||
alt="Standard vs Integration"> | alt="Standard vs Integration"> | ||
<figcaption style="max-width: 2400px; text-align: center;"> | <figcaption style="max-width: 2400px; text-align: center;"> | ||
− | Fig.8 - Standard vs Integration | + | Fig.8 - Standard vs Integration cassette. |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
<p style="margin-top: 1em;"> | <p style="margin-top: 1em;"> | ||
All terminators of the Marburg Collection were rebuild as "5a" parts similar to C-terminal tags. | All terminators of the Marburg Collection were rebuild as "5a" parts similar to C-terminal tags. | ||
− | This allowed to insert an antibiotic cassette at the position "5b". For this position | + | This allowed to insert an antibiotic cassette at the position "5b". For this position four |
+ | different | ||
antibiotic cassettes were designed.<br> | antibiotic cassettes were designed.<br> | ||
Our integration sites were also designed as connectors, so it is possible to build a gene cascade | Our integration sites were also designed as connectors, so it is possible to build a gene cascade | ||
− | with up to | + | with up to five genes that can be inserted into a single neutral site. All integration sites |
+ | function | ||
as 5'Con1 and 3'Con5 connectors, meaning they are always at the beginning of the first and the end | as 5'Con1 and 3'Con5 connectors, meaning they are always at the beginning of the first and the end | ||
− | of the last gene in a | + | of the last gene in a lvl2 construct.<br> |
It is important to note for the user that when designing the vector for integration, the origin | It is important to note for the user that when designing the vector for integration, the origin | ||
should not be compatible with the organism. This way, it enters the organism and then integrates | should not be compatible with the organism. This way, it enters the organism and then integrates | ||
Line 785: | Line 773: | ||
will be maintained in the transformed organism and it will be rather complicated to remove it. If | will be maintained in the transformed organism and it will be rather complicated to remove it. If | ||
there is no compatible origin available. We designed our toolbox so that it can always be | there is no compatible origin available. We designed our toolbox so that it can always be | ||
− | digested with NotI to linearize the integration cassette and extracted | + | digested with NotI to linearize the integration cassette and extracted from a gel. In a lot of |
− | cases transformations and homologous recombinations with linear DNA are a lot more efficient | + | cases transformations and homologous recombinations with linear DNA are a lot more efficient.<br> |
− | + | Our system offers the integration of up to five genes with four different selection markers at | |
− | Our system offers the integration of up to | + | five |
− | different integration sites. Therefore, the integration of up to | + | different integration sites. Therefore, the integration of up to twenty genes into the UTEX wild |
+ | type | ||
genome is possible. | genome is possible. | ||
</p> | </p> | ||
Line 802: | Line 791: | ||
class="lbl-toggle"> | class="lbl-toggle"> | ||
Fluorescence reporters for characterization of parts | Fluorescence reporters for characterization of parts | ||
+ | <hr style="width: unset;"> | ||
</label> | </label> | ||
<div class="collapsible-content"> | <div class="collapsible-content"> | ||
Line 808: | Line 798: | ||
When working in Synthetic Biology, reporter genes such as fluorescence proteins are indispensable | When working in Synthetic Biology, reporter genes such as fluorescence proteins are indispensable | ||
elements to characterize BioBricks. For a good characterization a suitable reporter is required. | elements to characterize BioBricks. For a good characterization a suitable reporter is required. | ||
− | But reporters can be more than just merely a detection tool for transcriptional activity but | + | But reporters can be more than just merely a detection tool for transcriptional activity but can |
− | + | also give a deeper insight into cellular conditions beyond the genetic context. We provide a | |
diverse set of reporters not only for the purpose of describing genetic tools but also for the | diverse set of reporters not only for the purpose of describing genetic tools but also for the | ||
sensing of a variety of parameters which are crucial for cyanobacteria. | sensing of a variety of parameters which are crucial for cyanobacteria. | ||
Line 858: | Line 848: | ||
<i>Source: FP Base (EYFP)</i> | <i>Source: FP Base (EYFP)</i> | ||
<p style="margin-top: 1em;"> | <p style="margin-top: 1em;"> | ||
− | eYFP is the mutant of green fluorescent protein naturally occuring in Aequorea victoria. It is a | + | eYFP is the mutant of green fluorescent protein naturally occuring in <i>Aequorea victoria</i>. |
+ | It is a | ||
preferred reporter for cyanobacteria as it bypasses the wavelength at which absorption | preferred reporter for cyanobacteria as it bypasses the wavelength at which absorption | ||
photoactive pigments occurs, resulting in stronger signal overall | photoactive pigments occurs, resulting in stronger signal overall | ||
Line 978: | Line 969: | ||
virtually no bleed-through of signal, making it suitable for dual fluorescent protein | virtually no bleed-through of signal, making it suitable for dual fluorescent protein | ||
applications like terminator characterization (see | applications like terminator characterization (see | ||
− | <a href="https://2019.igem.org/Team:Marburg/Composite_Part">here</a>). | + | <a href="https://2019.igem.org/Team:Marburg/Composite_Part" |
+ | target="_blank">here</a>). | ||
</p> | </p> | ||
</div> | </div> | ||
Line 986: | Line 978: | ||
<p> | <p> | ||
NanoLuc is a small luminescent reporter with just a molecular weight of 19,5 kDA. This reporter | NanoLuc is a small luminescent reporter with just a molecular weight of 19,5 kDA. This reporter | ||
− | stands out with a signal strength that is orders of magnitude higher | + | stands out with a signal strength that is orders of magnitude higher compared to traditional |
luminescent reporters. It is a very small protein and unlike the lux operon it is only a single | luminescent reporters. It is a very small protein and unlike the lux operon it is only a single | ||
− | gene, reducing the metabolic burden | + | gene, reducing the metabolic burden to the host to a bare minimum. Additionally it is not using |
ATP as a substrate which is a valuable energy resource in cells. This way it does not affect the | ATP as a substrate which is a valuable energy resource in cells. This way it does not affect the | ||
cellular context and acts as a truly orthogonal reporter. | cellular context and acts as a truly orthogonal reporter. | ||
Line 996: | Line 988: | ||
</p> | </p> | ||
<p> | <p> | ||
− | + | teLuc is a triple mutant of NanoLuc. Thanks to a modified substrate binding pocket it is able to | |
use DTZ as a substrate, resulting in a (42 nm) red-shift (from 460 nm to 502 nm peak) of | use DTZ as a substrate, resulting in a (42 nm) red-shift (from 460 nm to 502 nm peak) of | ||
emission. This bypasses the absorption of Chlorophyll A, making it the more suitable reporter for | emission. This bypasses the absorption of Chlorophyll A, making it the more suitable reporter for | ||
Line 1,005: | Line 997: | ||
</p> | </p> | ||
<p> | <p> | ||
− | Antares2 is a coupled bioluminescence protein consisting of | + | Antares2 is a coupled bioluminescence protein consisting of teLuc and two flanking CyOFP |
fluorescence reporters. It abuses the Bioluminescence Resonance Energy Transfer (BRET) to excite | fluorescence reporters. It abuses the Bioluminescence Resonance Energy Transfer (BRET) to excite | ||
CyOFP with the luminescence of TeLuc. This results in a further red-shift, making it suitable for | CyOFP with the luminescence of TeLuc. This results in a further red-shift, making it suitable for | ||
Line 1,014: | Line 1,006: | ||
detection of cellular conditions only fluorescent reporters are established yet. We present | detection of cellular conditions only fluorescent reporters are established yet. We present | ||
reporters for the two most important chemical parameters in cyanobacteria: pH and redox status. We | reporters for the two most important chemical parameters in cyanobacteria: pH and redox status. We | ||
− | saw that the pH of the media has a significant impact on the growth of the culture | + | saw that the pH of the media has a significant impact on the growth of the culture, which is |
− | + | ||
previously described <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC216614/">(Kallas, | previously described <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC216614/">(Kallas, | ||
− | + | Castenholz <i>et al.</i>, 1982)</a>. Cyanobacteria are not equipped to regulate their internal | |
+ | pH very well, | ||
yet they still depend on a stable proton gradient to keep up their photosynthetic machinery | yet they still depend on a stable proton gradient to keep up their photosynthetic machinery | ||
− | <a href="https://jb.asm.org/content/190/19/6318">(Billini et al.)</a>. We present | + | <a href="https://jb.asm.org/content/190/19/6318">(Billini <i>et al.</i>, 2008)</a>. We present |
+ | pHluorin2, a | ||
reporter that is modulated in its excitation peak by varying ph values. | reporter that is modulated in its excitation peak by varying ph values. | ||
</p> | </p> | ||
<div style="margin-top: 1em;"> | <div style="margin-top: 1em;"> | ||
<p> | <p> | ||
− | <b> | + | <b>pHlourin2 (S.e.)</b> |
</p> | </p> | ||
<table> | <table> | ||
Line 1,043: | Line 1,036: | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | <i>Source: FP Base ( | + | <i>Source: FP Base (pHluorin2)</i> |
<p style="margin-top: 1em;"> | <p style="margin-top: 1em;"> | ||
− | + | pHluorin2 is a mutant of GFP2. Its excitation maximum depends on the surrounding pH value. | |
Therefore it can be used to detect changes in the cellular pH. As described above a biosensor | Therefore it can be used to detect changes in the cellular pH. As described above a biosensor | ||
for this parameter could be of great use, especially in cyanobacteria. | for this parameter could be of great use, especially in cyanobacteria. | ||
− | <a href="">(Mahon, 2011)</a><br> | + | <a href="https://file.scirp.org/pdf/ABB20110300005_52257288.pdf" |
+ | target="_blank">(Mahon, 2011)</a><br> | ||
Another important cellular factor is the internal redox status. During photosynthesis reactive | Another important cellular factor is the internal redox status. During photosynthesis reactive | ||
oxygen species (ROS) are constantly produced as a byproduct. A critical mass of reactive oxygen | oxygen species (ROS) are constantly produced as a byproduct. A critical mass of reactive oxygen | ||
Line 1,056: | Line 1,050: | ||
For example, the overexpression of orthogonal thioredoxin peroxidase leads to the degradation | For example, the overexpression of orthogonal thioredoxin peroxidase leads to the degradation | ||
of ROS resulting in enhanced growth of PCC7942, | of ROS resulting in enhanced growth of PCC7942, | ||
− | <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297720/">(Kim et al.)</a> We present | + | <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297720/">(Kim <i>et al.</i>, 2018)</a> We |
+ | present | ||
rxYFP, a redox-sensitive reporter for cyanobacteria. | rxYFP, a redox-sensitive reporter for cyanobacteria. | ||
</p> | </p> | ||
Line 1,106: | Line 1,101: | ||
<i>Source: FP Base (sYFP)</i> | <i>Source: FP Base (sYFP)</i> | ||
<p style="margin-top: 1em;"> | <p style="margin-top: 1em;"> | ||
− | rxYFP is a redox-sensitive yellow fluorescent protein deriving from Aequorea victoria GFP. This | + | rxYFP is a redox-sensitive yellow fluorescent protein deriving from <i>Aequorea victoria</i> |
+ | GFP. This | ||
reporter contains a pair of redox-active Cys residues (Cys149 and Cys202), which are connected | reporter contains a pair of redox-active Cys residues (Cys149 and Cys202), which are connected | ||
through a disulphide bond under oxidative conditions, resulting in a 2.2-fold reduction of the | through a disulphide bond under oxidative conditions, resulting in a 2.2-fold reduction of the | ||
Line 1,121: | Line 1,117: | ||
</div> | </div> | ||
</section> | </section> | ||
+ | </div> | ||
</div> | </div> | ||
</html> | </html> | ||
{{Marburg/footer}} | {{Marburg/footer}} |
Latest revision as of 01:37, 14 December 2019
D E S I G N
"Always plan ahead. It wasn’t raining when Noah build the ark." - Richard Cushing
What does expanding the Golden Gate based Marburg Collection, automating time consuming lab work and
establishing the CRISPR/Cas12a system in Synechococcus elongatus UTEX 2973 have in common?
To achieve these objectives, it is always necessary to have a comprehensive theoretical preparation. It all
starts with literature research, summarizing the current state of the art and based on this developing own
ideas. To have the theoretical background settled, before the lab work starts, is a key point of every project
and
consumes many hours.
Because in the near future phototrophic organisms will be more and more relevant for biotechnological
applications, we want to establish the use of Synechococcus elongatus as a phototrophic organism for
Synthetic Biology. Following the principles of Synthetic Biology to simplify the process of engineering of
biological systems, we set it our goal to establish Synechococcus elongatus UTEX 2973 as the fastest and
most accessible phototrophic chassis to date, providing it as a wind tunnel for phototrophic organisms with user
friendly and standardized workflows.
In order to achieve these goals, a lot of effort has been put into designing, building, testing, evaluating and
learning. Further, these steps had to be iterated over and over again to elaborate our standardized designs. By
providing you our theoretical background we want to give you an insight in our decision-making.
S T R A I N
E N G I N E E R I N G
T O O L B O X
Strain Engineering
As mentioned in our description,
Synechococcus elongatus UTEX 2973 is no longer naturally competent, presumably due to a
point mutation in the pilN gene (
Li
et
al., 2018), which means that when genetically engineering this organism other ways
to
introduce exogenous DNA have to be taken into consideration. This is mainly done through
electroporation or conjugation - especially triparental conjugation
(Yu et al., 2015). Triparental
conjugation into the UTEX 2973 strain is typically performed with two E. coli HB101
strains, one harboring the pRL443 plasmid and one harboring the pRL623 plasmid. The latter
strain is
then again transformed with the plasmid of interest, the prior is used as the conjugal strain -
both have to be incubated together with the cyanobacteria for the conjugation to take place
(
Wendt et al., 2016).
To overcome this time-consuming process, we planned to reintroduce natural competence into our
strain. It was already shown that this can be done by integrating an intact copy of the
pilN gene into one of the neutral sites
(Li
et
al., 2018), though this technique is not ideal: you have to add an antibiotic
cassette in
order to keep selective pressure on the bacteria, so that they integrate the new gene into every
chromosome copy. This antibiotic resistance will persist in the strain, meaning that when
further engineering the organism later on, this one resistance can not be used e.g. in vectors
for transient expression - a huge downside. Furthermore, one of the neutral sites has to be
used, resulting in a strain that has less neutral sites available for further introduction of
genes.
Although we did not prefer this method, we still tried, as we were not sure, if our other approach would prove to be successful. We also used extensive bioinformatic tools to identify new integration sites in UTEX 2973, which can be used if one were to reintroduce natural competence in the above mentioned way. Additionally, we came up with a plan to revert the point mutation in the pilN gene with a CRISPR/Cas12a system.
This approach is promising, as the integration of the new pilN copy only enabled a low
efficiency of natural transformation, which might be due to the point mutation negatively
affecting expression of the pilO and pilQ genes laying downstream of pilN
(Li et al., 2018 ;
Barten and
Lill, 1995). As CRISPR/Cas12a allows accurate targeting of genetic sequences, we designed
a
gRNA leading the Cas12a protein to the pilN locus. The repair template was taken from
the S. elongatus PCC 7942 genome, where the gene is still intact, allowing the cell to
repair the cut introduced by Cas12a accordingly, reversing the point mutation, which leads to an
intact copy of pilN again - a more elegant approach than simply inserting a second copy
of the gene. As our own CRISPR system was still under construction at that point, we had to rely
on
pSL2680, a replicating base vector for constructing CRISPR/Cas12a editing plasmids by
Ungerer and Pakrasi, 2016.
We followed their protocol (available here on
Addgene), annealing oligos to construct the gRNA. Small overhangs were added to enable
the
ligation into the AarI-digested vector, where a lacZ cassette was replaced, which allowed
for blue/white screening of recombinant colonies. Additionally, the repair template had to be
constructed by PCR with added overhangs for
the following Gibson reaction. As stated, it was taken from the S. elongatus PCC 7942
genome. It was designed in such a way that the point mutation inside the UTEX 2973 genome was
part of the PAM sequence for Cas12a, meaning that the repair template did not include the PAM
and would not be cut by the enzyme.
![blub](https://static.igem.org/mediawiki/2019/a/a3/T--Marburg--UDAR-PCC-UDAR-rep.png)
After the successful Gibson assembly of gRNA and repair template into the Cas12a carrying vector, nearly two weeks had passed, indicating that working with this vector can be quite tedious and time consuming. This is one of the many reasons why we chose to implement such a CRISPR system into our MoClo based toolbox. While building this system we made sure to directly prove it by using it to reverse this point mutation, making sure that we tackle this crucial goal through multiple approaches.
CRISPR/Cas systems are powerful tools that have gained a lot of popularity in recent years.
As they can be used for a wide array of applications - like the integration of whole genes,
alternation of single nucleotides, knock-outs of whole genetic regions, as well as the use of the
DNA-binding property in a multitude of applications through so called deadCas systems, where the
Cas protein does not exhibit nuclease activity
(Hsu et al.,
2014)
- we were eager to implement such a system into our own
toolbox. Diving into literature we
noticed many different systems are available, the most commonly used one being CRISPR/Cas9, and we
began to wonder which of them we should use.
In our description
we presented
CRISPR/Cas9 and CRISPR/Cas12a, showing the differences of these two systems. Looking deeper into
CRISPR/Cas12a we noticed a few advantages that finally led us to choose it as our preferred
system.
As the sgRNA used as a guide for Cas9 is usually ~100nt long, chemical synthesis is more complex
and expensive in comparison to the ~43nt needed for the Cas12a guiding crRNA
(Swarts and Jinek, 2018)
- an unpleasant fact, especially for iGEM teams that do not have many resources available to them,
but the main reasons we chose Cas12a are others. Multiplexed gene editing is one of the key
features of these CRISPR/Cas systems, but how to actually apply it differs:
For Cas9 each sgRNA is in need of its own promoter, which means that they have to be expressed
from different vectors or a multi cassette vector (Swarts &
Jinek, 2018;
Z. Zhang
et al., 2016). In contrary, multiplexed genome editing with Cas12a can be achieved
simply
by expressing all of the needed guide RNAs in one transcriptional unit, the crRNA.
This is a huge advantage of Cas12a. Furthermore,
CRISPR/Cas9 was shown to be toxic in cyanobacteria
(Wendt et al., 2016), which is one of the foremost reasons CRISPR technologies have
not been
widely applied in cyanobacteria - the usage of Cas12a though, seems to be less
toxic (Ungerer and Pakrasi, 2016),
making it the ideal candidate for the Green Extension of the
Marburg Collection.
The actual implementation of the CRISPR/Cas12a system into our toolbox necessitated a well thought
out plan. The design of our CRISPR/Cas12a system was mainly affected by the fact that we wanted to
have a convenient and rapid tool for genomic manipulation. The lvl 0 part of the
Cas12a protein was created via PCR amplification from the plasmid pSL2680, but special overhangs
were added in order to clone the PCR product into a lvl 0 acceptor vector. The part was introduced
as a coding sequence (CDS) part in the MoClo standard to be included in the Green Expansion of the
Marburg Collection. The lvl 1 part of the Cas12a protein was equipped with a rather weak promoter
so that the toxicity caused by overproduction of the endonuclease could be kept low. The parts
used for lvl 1 assembly were: pMC0_1_03 + pMC0_2_03 + pMC0_3_07 + pMC0_4_33 + pMC0_5_07 +
pMC0_6_17. For the construction of the crRNA part the design of the plasmid pSL2680 was mainly
maintained, but the lacZ cassette was replaced by a GFP cassette with BsmBI cutting sites
to enable easier screening
of gRNA assembly and to reduce expenses for X-Gal/IPTG. It was constructed as a part reaching
from the RBS site to the end of the terminator site. As the whole system is built for modular
cloning in PhytoBrick syntax, it is possible to freely exchange the parts around the Cas12a and
crRNA parts - in this way the amount of crRNA/Cas12a can be controlled by choosing promoters with
different strengths.
Our initial plan was to synthesize the crRNA with the desired overhangs, but as the sequence
contains multiple direct repeats, it was not possible for providers to synthesize this construct,
which is why we split it into four different parts that then had to be assembled. For this
assembly the four parts were first cloned into the pJET1.2/blunt vector by Thermo Scientific and
then digested with BsaI while the acceptor vector was digested with BsmBI. In this way the final
vector still contains BsaI recognition sites, so that it can be used in a level 1 assembly Golden
Gate reaction. The cloning of the level 2 part with this crRNA part was done by ending with a
ligation step to make sure the GFP dropout remains in the vector.
Following our dream to create the most versatile, MoClo compatible shuttle vector for
cyanobacteria we made sure to pay attention to detail. When creating new shuttle vectors, one of
the most important points to consider is the replication element that is being used, mainly due
to the wanted copy number and a phenomenon called plasmid incompatibility. Plasmids harboring
the same replication or partitioning system can often not be stably maintained in a cell - they
are incompatible (Novick &
Hoppenstaedt, 1978). With multiple different plasmids bearing the same replication
elements,
the replication machinery will randomly choose which plasmids to replicate, leading to one of
the different plasmids being copied more frequently than the other
. As we used the minimal replication element from the pANS plasmid
of S. elongatus in our shuttle-vectors, in order to have a native origin of replication,
we had to consider such plasmid incompatibilities and made sure to
cure our strain of
the endogenous pANS
- which we could successfully proove.
The next step was the creation of our own, modular shuttle vector. For this we had to pay
attention, as we had to fit it to the PhytoBrick standard in order to use it in our Green
Extension of the Marburg Collection. This means that we had to remove some restriction enzyme
cutting sites at multiple points in the sequence: In repB and repA - both a CDS of the minimal
replication element needed for the vector - lay one BsaI recognition site each, which were
removed by introducing a silent point mutation. This point mutation, in both cases, introduced a
synonym codon for glutamic acid, so they should not cause any issues later on.
A BsmBI site was found within the non-coding sequence of the minimal replication element,
meaning that this had to be changed with a more careful approach, as any change could have heavy
influence on secondary structure and potentially impair the function. Due to this reason we made
sure to try all possible variants of mutations to remove the recognition site of the restriction
enzyme.
In order to assemble our desired part we synthesized different parts of it with the mutations we
introduced. Due to the length and complexity of the sequence we had to divide the synthesis of
the minimal replication element into three parts that later had to be fused together.
Additionally, we wanted to implement a reporter for easy selection. We chose RFP, which was
amplified out of the Lvl0_8_Amp/ColE1 part from last years Marburg Collection in addition with
the ColE1 ori that can be found on it. This means that our vector does not just contain the
cyanobacterial ori of our strain, but also a high copy origin for replication in E. coli
(Gerhart et al., 2002). As an
antibiotic cassette we chose spectinomycin, which we also amplified by PCR, this time from
pAM4787
(Chen
et al., 2016). Finally, those five fragments - the three parts of the minimal
replication
element, the ColE1 ori & RFP cassette and the spectinomycin resistance cassette (aadA) - were
fused together in a Gibson reaction, resulting in BBa_K3228069 (sometimes also called lvl 1
ori), the first cyanobacterial shuttle vector for cloning lvl 1 constructs in a modular way.
This part has two BsaI sites that flank the RFP cassette, so that this genetic element will be
exchanged with the other parts in a lvl 1 Golden Gate reaction.
In addition to this, we created a second shuttle vector, this time for cloning lvl 2 constructs: This vector has mostly the same design as BBa_K3228069, but the RFP cassette is flanked by BsmBI sites, enabling the construction of lvl 2 vectors.
Furthermore this part bears a kanamycin resistance cassette instead of the spectinomycin resistance of the lvl 1 ori. This part was assembled in a four part Gibson reaction, as in addition to ColE1 and the RFP cassette also the kanamycin resistance cassette could be amplified via PCR, in this case from pYTK_0_84, a plasmid from the Dueber MoClo Yeast Toolkit, resulting in BBa_K3228089 (sometimes called lvl 2 ori). For all these cloning processes special overhangs had to be added for Gibson Assembly.
Toolbox
The Marburg Collection is a toolbox from last year’s iGEM Marburg team for the rational design of metabolic pathways and genetic circuits or any other DNA construct. Thanks to its flexible design based on the ‘Dueber toolbox’ design from Lee et al. (2015) it can be used in a multitude of chassis: since it complies with the PhytoBrick standard, it can even be extended to eukaryotic chassis such as plants. The design of the toolbox is rather simple and user friendly: lvl 0 parts are the basic foundation of every assembly. They contain a single genetic element such as a promoter or terminator. Up to 8 lvl 0 parts are used to build a lvl1 plasmid containing a single transcription unit. Up to 5 of these transcription units can be assembled together in a lvl 2 plasmid (Lee et al., 2015).
Here we present a new feature of the Marburg Collection 2.0: placeholders. These parts make it
possible to construct plasmids with a placeholder, which can be later on exchanged with any part
of the same type.
A key feature in our expansion is the addition of placeholders that allow high throughput assembly
of plasmids that only differ in one part. A promoter placeholder for example is built into a lvl 1
construct at the promoter position. Instead of a promoter however it contains a GFP cassette and
reversed BsaI cutting sites. This allows BsaI cleavage and removal of the GFP cassette even after
assembly, due to the fact that the BsaI recognition site is not removed from the placeholder.
After that, any promoter of choice can be inserted at that position. After ligation, no BsaI cutting sites remain on the vector, so in the end mainly the newly assembled remain. These steps also happen in a one pot one step reaction just like any other Golden Gate assembly.
White/green selection under UV light can be used to determine the colonies with the right plasmid: green ones still contain the plasmid with a placeholder, white ones contain the desired vector.
Placeholders exist for every position from 1-6, but technically placeholders can also span multiple positions to insert multiple parts at once. For example a placeholder for the position promoter and RBS could be replaced with any combination of promoter and RBS that is deemed right for a specific application. This however would then be a three part assembly. The application of such parts is so narrow that we decided to build the most useful ones. Thanks to its clever design the construction of more placeholder is so simple that it can be done by the user itself with a single site directed mutagenesis of a flank.
The heart piece of the Green Expansion is BBa_K3228069, a lvl 0 part containing origins of replication for E. coli and S. elongatus as well as a spectinomycin cassette. It resembles a type 7+8 (antibiotic cassette + ori) composite part and can be seen as the cyanobacteria specific lvl1 entry vector. Another version of this entry vector contains a kanamycin cassette and BsmbI cutting sites and can be used as the lvl2 entry vector. Just like in our lvl0 entry vectors for basic parts, we prompted for a fluorescence based reporter in the dropout, rather than lacZ for blue/white screening. Therefore both vectors contain an RFP dropout to signal an insertion. Using this vector in our updated Golden Gate assembly protocols, we achieve a rate of about 9:1 white to red colonies, showing that the assembly is rather efficient.
Here we present the design of the plasmids and the workflow used to characterize BioBricks.
In order to characterize BioBricks they need to be inserted into a measurement vector that is
stably maintained in cyanobacteria. The design of the plasmids to characterize our parts was an
amazing experience as it was one of the first times that we acted not only as creators but also as
users of our toolbox. Therefore design of the workflow and design of new parts was tied together
very closely.
The criteria that the measurement vectors need to meet are some of the most basic
principles of Synthetic Biology:
In order to be comparable, all of the constructs must be
almost identical and only differ in the part to be tested. Instead of building each construct
independently we utilized our placeholders to build all
measurement plasmids for the same type of part from the same blueprint.
We present a set of
measurement entry vectors for the characterization of BioBricks in cyanobacteria (Part range
BBa_K3228073 to BBa_K3228075 as well as BBa_K3228090). They contain our MoClo compatible shuttle
vector for cyanobacteria BBa_K3228069 and are therefore the only MoClo based vector for the
characterization of BioBricks in cyanobacteria. These pre assembled lvl1 plasmids contain a
placeholder for their respective BioBrick type that acts as a dropout to quickly and effortlessly
insert any part of the same type for an easy characterization. In our results we show how these
measurement entry vectors can save a lot of effort and money when characterizing a greater library
of parts. Additionally, the usage of the same entry vector for each measurement will aid in
greater comparability and reproducibility.
For greater comparability across other data sets we decided to use similar BioBricks as in
measuring the toolbox for Vibrio natriegens in the last year. The design from there on was
pretty straight forward for promoter and RBS.
For terminators however the design is a bit more intricate: a terminator is not measured in its
activity but rather in its isolative power. Hence, a strong terminator should result in a weak
signal. On top of that, measuring the activity both upstream and downstream of the terminator with
two independent reporters would give insight on the exact transcriptional activity around the area
of the terminator (Chen et al.,
2013),
resulting in the most accurate results in respect to the molecular dynamics of a terminator
(see modeling).
A lvl2 plasmid was logically the easiest way to construct such a part. We designed a normal lvl1
plasmid containing an mTurqouise reporter and a secondary lvl1 plasmid containing an YFP reporter
but missing a promoter.
The fraction of the signal strength of YFP and mTurquoise describe the isolative capacity of the
terminator best (Chen et al.,
2013).
This way of calculating isolative strength is also used in RNA-seq to determine the strength of
terminators.
Artificial neutral integration site options (aNSo) for our purpose in Synechococcus
elongatus needed to fulfill three criteria, to be genuinely considered as potential
candidates.
A highly precise algorithm was implemented in a Python script to find these potential candidates
(see modeling) by describing the following
criteria. First, no gene and transcription start site (TSS), i.e. no CDS, was allowed to be
disturbed, assuring that no lethal modification was created by integration. Thereby, we searched
for intergenic regions where no TSS had been identified, with a length of at least 500 bp. These
sequences had to be extended in both 3’ and 5’ direction up to a length of at least 2500 bp
providing flanks to ensure the integration by homologous recombination, which should be performed
in the lab subsequently. In the middle of these sequences any gene of interest can be inserted,
which gets integrated into the genome by the mentioned homologous recombination, due to homologous
flanks. Second, integration site sequences were not allowed to contain restriction sites that
interfere with the iGEM standards to simplify the cloning process and make them more cross
compatible. All sequences that contained such restriction site were discarded. Executing this
newly developed and unique algorithm resulted in two unique aNSo's within the genome of S.
elongatus.
For a successful homologous integration the sequence to be integrated needs to be flanked by two
integration sites homologous to the neutral site on the target genome. Additionally, the
integrated sequence needs to contain an appropriate selection marker to be able to select for
integration events.
It is included in the syntax of the Marburg Collection, that the positions 1 and 6 can not only be
used for connectors but for integration sites as well. Since integration sites contain a BsmBI
restriction site just like a connector part, their construction is a bit more intricate than a
normal part:
Building a homology/ connector part
-
Step 1: Find your integration site.
For more on this see Modeling: integration sites -
Step 2: Determine your two homology sequence. Optimally the two sequences should span
around 800-1200 and not begin or end in an ORF. Leave 40 bp of space in a region without an
ORF between the two sequences, this increases the likelihood for successful recombination
events.
Note that these bases will be knocked out in the recombination event. - Step 3: Amplify both integration sites via a genomic PCR using the overhang primers for 5’Connectors (upstream homology sequence) and 3’Connectors (downstream homology sequence), respectively. Check if your PCR worked with a test agarose gel.
- Step 4: Purify your PCR sample using any commercial kit to remove genomic DNA.
- Step 5: Digest your PCR sample with BsaI (Note that this is uncommon for LVL 0 Cloning but necessary because of the internal BsmBI restriction site)
- Step 6: Digest your lvl0 Entry vector with BsmBI and purify it over an agarose gel to remove the GfP Dropout.
- Step 7: Ligate your digested PCR sample and lvl0 Entry vector overnight.
- Step 8: Transform your ligation as usual in an E. coli or V. natriegens strain for cloning. Thanks to the predigested lvl0 entry vector most colonies should appear white. Pick a few colonies and verify the construct inside by sequencing. Usually at least 1 in 2 sequencing results yields the correct construct.
In a lvl1 construct, the positions 2-5 representing a full transcription unit (promoter, RBS, CDS, terminator) would be integrated into the genome, while positions 7-8 (origin of replication, antibiotic cassette) would be cut off in the recombination event. The issue with this assembly would be that a marker for the selection after integration is completely missing. Hence, we decided to split the position of the terminator in a similar fashion in which C-terminal tags were integrated into the syntax last year:
All terminators of the Marburg Collection were rebuild as "5a" parts similar to C-terminal tags.
This allowed to insert an antibiotic cassette at the position "5b". For this position four
different
antibiotic cassettes were designed.
Our integration sites were also designed as connectors, so it is possible to build a gene cascade
with up to five genes that can be inserted into a single neutral site. All integration sites
function
as 5'Con1 and 3'Con5 connectors, meaning they are always at the beginning of the first and the end
of the last gene in a lvl2 construct.
It is important to note for the user that when designing the vector for integration, the origin
should not be compatible with the organism. This way, it enters the organism and then integrates
into the genome or disappears as it cannot be replicated in its new host. Otherwise the vector
will be maintained in the transformed organism and it will be rather complicated to remove it. If
there is no compatible origin available. We designed our toolbox so that it can always be
digested with NotI to linearize the integration cassette and extracted from a gel. In a lot of
cases transformations and homologous recombinations with linear DNA are a lot more efficient.
Our system offers the integration of up to five genes with four different selection markers at
five
different integration sites. Therefore, the integration of up to twenty genes into the UTEX wild
type
genome is possible.
When working in Synthetic Biology, reporter genes such as fluorescence proteins are indispensable elements to characterize BioBricks. For a good characterization a suitable reporter is required. But reporters can be more than just merely a detection tool for transcriptional activity but can also give a deeper insight into cellular conditions beyond the genetic context. We provide a diverse set of reporters not only for the purpose of describing genetic tools but also for the sensing of a variety of parameters which are crucial for cyanobacteria.
eYFP
Aequorea victoria | |
Excitation Maximum (nm) | 515 |
Emission Maximum (nm) | 527 |
Extinction Coefficient (M-1 cm-1) | 67,000 |
Quantum Yield | 0.67 |
Brightness | 44.89 |
pKa | 6.9 |
Maturation (min) | 9.0 |
Life- span (ns) | 3.1 |
eYFP is the mutant of green fluorescent protein naturally occuring in Aequorea victoria. It is a preferred reporter for cyanobacteria as it bypasses the wavelength at which absorption photoactive pigments occurs, resulting in stronger signal overall (Kukolka & M. Niemeyer, 2004).
![Graph](https://static.igem.org/mediawiki/2019/b/b6/T--Marburg--Reporter--UTEX-Spectra.png)
Additionally, autofluorescence of cyanobacterial cells is rather low at that point, resulting in a stronger signal compared to the background, increasing the resolution of characterizations.
sYFP2 (S.e.)
Aequorea victoria | |
Excitation Maximum (nm) | 515 |
Emission Maximum (nm) | 527 |
Extinction Coefficient (M-1 cm-1) | 101,000 |
Quantum Yield | 0.68 |
Brightness | 68.68 |
pKa | 6.0 |
Maturation (min) | 4.1 |
Life- span (ns) | 2.9 |
sYFP is a superfolded version of YFP. Thanks to faster maturation it leads not only to a twofold signal strength compared to eYFP: the fast maturation also ensures that every transcribed mRNA leads to the same amount of correctly folded fluorescent protein. This makes measurements more robust towards varying cellular contexts.
mTurquoise2 (S.e.)
Aequorea victoria | |
Excitation Maximum (nm) | 434 |
Emission Maximum (nm) | 474 |
Extinction Coefficient (M-1 cm-1) | 30,000 |
Quantum Yield | 0.84 |
Brightness | 25.2 |
pKa | 4.5 |
Maturation (min) | 112.2 |
Life- span (ns) | 3.7 |
mTurquoise2 is a brighter fluorescent variant of mTurquoise with faster maturation and a high photostability, making it one of the better for microscopy applications. Thanks to a shifted emission maximum it is possible to detect both, YFP and mTurquoise in single cells with virtually no bleed-through of signal, making it suitable for dual fluorescent protein applications like terminator characterization (see here).
NanoLuc
NanoLuc is a small luminescent reporter with just a molecular weight of 19,5 kDA. This reporter stands out with a signal strength that is orders of magnitude higher compared to traditional luminescent reporters. It is a very small protein and unlike the lux operon it is only a single gene, reducing the metabolic burden to the host to a bare minimum. Additionally it is not using ATP as a substrate which is a valuable energy resource in cells. This way it does not affect the cellular context and acts as a truly orthogonal reporter.
TeLuc
teLuc is a triple mutant of NanoLuc. Thanks to a modified substrate binding pocket it is able to use DTZ as a substrate, resulting in a (42 nm) red-shift (from 460 nm to 502 nm peak) of emission. This bypasses the absorption of Chlorophyll A, making it the more suitable reporter for phototrophic organism.
Antares2
Antares2 is a coupled bioluminescence protein consisting of teLuc and two flanking CyOFP
fluorescence reporters. It abuses the Bioluminescence Resonance Energy Transfer (BRET) to excite
CyOFP with the luminescence of TeLuc. This results in a further red-shift, making it suitable for
applications like deep tissue analysis. Additionally, it can be used in conjunction with NanoLuc
thanks to the utilization of two distinct substrates as well as varying emission peaks. Therefore
it is the world’s only dual luminescent detector pair.
Luminescence is a great tool for accurate measurements, but in the world of biosensors for the
detection of cellular conditions only fluorescent reporters are established yet. We present
reporters for the two most important chemical parameters in cyanobacteria: pH and redox status. We
saw that the pH of the media has a significant impact on the growth of the culture, which is
previously described (Kallas,
Castenholz et al., 1982). Cyanobacteria are not equipped to regulate their internal
pH very well,
yet they still depend on a stable proton gradient to keep up their photosynthetic machinery
(Billini et al., 2008). We present
pHluorin2, a
reporter that is modulated in its excitation peak by varying ph values.
pHlourin2 (S.e.)
Aequorea victoria | acidic (pH 5,5) | alkaline (pH 7,5) |
Excitation Maximum (nm) | 395 | 475 |
Emission Maximum (nm) | 509 | 509 |
pHluorin2 is a mutant of GFP2. Its excitation maximum depends on the surrounding pH value.
Therefore it can be used to detect changes in the cellular pH. As described above a biosensor
for this parameter could be of great use, especially in cyanobacteria.
(Mahon, 2011)
Another important cellular factor is the internal redox status. During photosynthesis reactive
oxygen species (ROS) are constantly produced as a byproduct. A critical mass of reactive oxygen
species leads to serious cell damage and cell toxicity through chemical alterations of proteins,
DNA and lipids. Especially under high light conditions the redox status becomes a crucial
parameter as it can threaten the cellular fitness.
For example, the overexpression of orthogonal thioredoxin peroxidase leads to the degradation
of ROS resulting in enhanced growth of PCC7942,
(Kim et al., 2018) We
present
rxYFP, a redox-sensitive reporter for cyanobacteria.
rxYFP (S.e.)
Aequorea victoria | |
Excitation Maximum (nm) | 515 |
Emission Maximum (nm) | 527 |
Extinction Coefficient (M-1 cm-1) | 101,000 |
Quantum Yield | 0.68 |
Brightness | 68.68 |
pKa | 6.0 |
Maturation (min) | 4.1 |
Life- span (ns) | 2.9 |
rxYFP is a redox-sensitive yellow fluorescent protein deriving from Aequorea victoria GFP. This reporter contains a pair of redox-active Cys residues (Cys149 and Cys202), which are connected through a disulphide bond under oxidative conditions, resulting in a 2.2-fold reduction of the emission peak. This allows to determine the redox potential in the environment which then expressed the output of fluorescence.