Difference between revisions of "Team:Marburg/Design"

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As mentioned in our <a href="https://2019.igem.org/Team:Marburg/Description">description</a>,
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<p>
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<i>Synechococcus elongatus </i>UTEX 2973 is no longer naturally competent, presumably due to a
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As mentioned in our <a href="https://2019.igem.org/Team:Marburg/Description">description</a>,
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point mutation in the pilN gene (
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<i>Synechococcus elongatus </i>UTEX 2973 is no longer naturally competent, presumably due to a
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<a href=https://www.sciencedirect.com/science/article/pii/S1096717618301757?via%3Dihub> Li et al.,
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point mutation in the pilN gene (
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2018</a>), which means that when genetically engineering this organism other ways to introduce
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<a href="https://www.sciencedirect.com/science/article/pii/S1096717618301757?via%3Dihub"> Li et
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exogenous DNA have to be taken into consideration. This is mainly done through electroporation or
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al., 2018</a>), which means that when genetically engineering this organism other ways to
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conjugation - especially triparental conjugation
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introduce exogenous DNA have to be taken into consideration. This is mainly done through
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<a href=https://www.nature.com/articles/srep08132>(Yu et al., 2015)</a>. Triparental conjugation
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electroporation or conjugation - especially triparental conjugation
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into the UTEX 2973 strain is typically performed with two <i>E. coli</i> HB101 strains, one
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<a href="https://www.nature.com/articles/srep08132">(Yu et al., 2015)</a>. Triparental
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harboring the pRL443 and one harboring the pRL623 plasmid. The latter strain is then again
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conjugation into the UTEX 2973 strain is typically performed with two <i>E. coli</i> HB101
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transformed with the plasmid of interest, the prior is used as the conjugal strain - both have to
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strains, one harboring the pRL443 and one harboring the pRL623 plasmid. The latter strain is
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be incubated together with the cyanobacteria for the conjugation to take place
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then again transformed with the plasmid of interest, the prior is used as the conjugal strain -
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<a href=https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0514-7> (
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both have to be incubated together with the cyanobacteria for the conjugation to take place
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Wendt et al., 2016)</a><br>
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<a href="https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0514-7">(
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To overcome this time-consuming process, we planned to reintroduce natural competence into our
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Wendt et al., 2016)</a><br>
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strain. It was already shown, that this can be done by integrating an intact copy of the
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To overcome this time-consuming process, we planned to reintroduce natural competence into our
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<i>pilN</i> gene into one of the neutral sites
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strain. It was already shown, that this can be done by integrating an intact copy of the
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<a href=https://www.sciencedirect.com/science/article/pii/S1096717618301757?via%3Dihub> (Li et al.
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<i>pilN</i> gene into one of the neutral sites
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, 2018)</a>, though this technique is not ideal: you have to add an antibiotic cassette in order
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<a href="https://www.sciencedirect.com/science/article/pii/S1096717618301757?via%3Dihub"> (Li et
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to keep selective pressure on the bacteria, so that they integrate the new gene into all the
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al., 2018)</a>, though this technique is not ideal: you have to add an antibiotic cassette in
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chromosome copies. This antibiotic resistance will persist in the strain, meaning that when
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order to keep selective pressure on the bacteria, so that they integrate the new gene into all
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further engineering the organism later on, this one resistance can not be used e.g. in vectors for
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the chromosome copies. This antibiotic resistance will persist in the strain, meaning that when
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transient expression - a huge downside. Furthermore, one of the neutral sites has to be used,
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further engineering the organism later on, this one resistance can not be used e.g. in vectors
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resulting in a strain that has less neutral sites available for further introduction of genes.<br>
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for transient expression - a huge downside. Furthermore, one of the neutral sites has to be
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<figure Style="text-align: center;">
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used, resulting in a strain that has less neutral sites available for further introduction of
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genes.
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</p>
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<figure style="text-align: center;">
 
<img style="height: 60ex; width: 90ex;"
 
<img style="height: 60ex; width: 90ex;"
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src=https://static.igem.org/mediawiki/2019/1/1d/T--Marburg--StrainEng_pilNns2.svg alt="NS bild">
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src="https://static.igem.org/mediawiki/2019/1/1d/T--Marburg--StrainEng_pilNns2.svg"
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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 et al. The pilN-Gene gets
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</figcaption>
 
</figcaption>
 
</figure>
 
</figure>
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Although we did not prefer this method, we still tried it, as we were not sure, if our other
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<p style="margin-top: 1em;">
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approach would prove to be successful. We also used extensive bioinformatic tools to identify
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Although we did not prefer this method, we still tried it, as we were not sure, if our other
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<a href="https://2019.igem.org/Team:Marburg/Parts">new integration sites</a> in UTEX 2973, which
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approach would prove to be successful. We also used extensive bioinformatic tools to identify
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can be used if one were to reintroduce natural competence in the above mentioned way.
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<a href="https://2019.igem.org/Team:Marburg/Parts">new integration sites</a> in UTEX 2973, which
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Additionally, we came up with a plan to revert the point mutation in the <i>pilN</i> gene with a
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can be used if one were to reintroduce natural competence in the above mentioned way.
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CRISPR/Cas12a system.
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Additionally, we came up with a plan to revert the point mutation in the <i>pilN</i> gene with a
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<figure Style="text-align:center">
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CRISPR/Cas12a system.
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</p>
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<figure style="text-align:center">
 
<img style="height: 80ex; width:80ex"
 
<img style="height: 80ex; width:80ex"
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src=https://static.igem.org/mediawiki/2019/8/8f/T--Marburg--StrainEng_pilNrepair.svg alt="Casa">
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src="https://static.igem.org/mediawiki/2019/8/8f/T--Marburg--StrainEng_pilNrepair.svg"
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alt="Casa">
 
<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
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</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
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<p style="margin-top: 1em;">
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efficiency of natural transformation, which might be due to the point mutation negatively
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This approach is promising, as the integration of the new <i>pilN</i> copy only enabled a low
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affecting expression of the <i>pil0</i> and <i>pilQ</i>genes laying downstream of <i>pilN</i>
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efficiency of natural transformation, which might be due to the point mutation negatively
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<a href=https://academic.oup.com/femsle/article/129/1/83/442013>(Li et al., 2018 ; Barten and
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affecting expression of the <i>pil0</i> and <i>pilQ</i>genes laying downstream of <i>pilN</i>
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Lill, 1995)</a>. As CRISPR/Cas12a allows accurate targeting of genetic sequences, we designed a
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<a href="https://academic.oup.com/femsle/article/129/1/83/442013">(Li et al., 2018 ; Barten and
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crRNA leading the Cas12a protein to the <i>pilN</i> locus. The repair template was taken from
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Lill, 1995)</a>. As CRISPR/Cas12a allows accurate targeting of genetic sequences, we designed a
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the <i>S. elongatus</i> PCC 7042 genome, where the gene is still intact, allowing the cell to
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crRNA leading the Cas12a protein to the <i>pilN</i> locus. The repair template was taken from
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repair the cut introduced by Cas12a accordingly, reversing the point mutation, which leads to an
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the <i>S. elongatus</i> PCC 7042 genome, where the gene is still intact, allowing the cell to
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intact copy of <i>pilN</i> again - a more elegant approach than simply inserting a second copy of
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repair the cut introduced by Cas12a accordingly, reversing the point mutation, which leads to an
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the gene. As our own CRISPR system was still in building at that point, we had to rely on pSL2680,
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intact copy of <i>pilN</i> again - a more elegant approach than simply inserting a second copy
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a replicating base vector for constructing CRISPR/Cas12a editing plasmids by
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of the gene. As our own CRISPR system was still in building at that point, we had to rely on
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<a href=https://www.nature.com/articles/srep39681>Ungerer and Pakrasi, 2016</a>.<br>
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pSL2680, a replicating base vector for constructing CRISPR/Cas12a editing plasmids by
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We followed their protocol <a href="https://www.addgene.org/85581">(available here on
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<a href="https://www.nature.com/articles/srep39681">Ungerer and Pakrasi, 2016</a>.<br>
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Addgene)</a>, annealing oligos to construct the crRNA. Small overhangs were added to enable the
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We followed their protocol <a href="https://www.addgene.org/85581">(available here on
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ligation into the AarI-digested vector, where a <i>lacZ</i> cassette was replaced, which allowed
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Addgene)</a>, annealing oligos to construct the crRNA. Small overhangs were added to enable the
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for blue/white screening of recombinant colonies. This cloning step alone took approximately a
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ligation into the AarI-digested vector, where a <i>lacZ</i> cassette was replaced, which allowed
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week. Additionally, the repair template had to be constructed by PCR with added overhangs for the
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for blue/white screening of recombinant colonies. This cloning step alone took approximately a
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following Gibson reaction. As stated, it was taken from the <i>S. elongatus</i> PCC 7942 genome.
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week. Additionally, the repair template had to be constructed by PCR with added overhangs for
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It was designed in such a way that the point mutation inside the UTEX 2973 genome was part of the
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the following Gibson reaction. As stated, it was taken from the <i>S. elongatus</i> PCC 7942
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PAM sequence for Cas12a, meaning that the repair template did not include the PAM and would not be
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genome. It was designed in such a way that the point mutation inside the UTEX 2973 genome was
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cut by the enzyme.
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part of the PAM sequence for Cas12a, meaning that the repair template did not include the PAM
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and would not be cut by the enzyme.
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</p>
 
<figure Style="text-align:center">
 
<figure Style="text-align:center">
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<img style="height: 60ex; width:120ex"
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<img style="height: 60ex; width: 120ex;"
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src=https://static.igem.org/mediawiki/2019/a/a3/T--Marburg--UDAR-PCC-UDAR-rep.png alt="blub">
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src="https://static.igem.org/mediawiki/2019/a/a3/T--Marburg--UDAR-PCC-UDAR-rep.png" alt="blub">
 
<figcaption>
 
<figcaption>
 
Fig.3:FigureX: The Top of the Figure shows a pars of the pilN gene of UTEX 2973. You can
 
Fig.3:FigureX: The Top of the Figure shows a pars of the pilN gene of UTEX 2973. You can
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</figure>
 
</figure>
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After the successful Gibson assembly of crRNA and repair template into the Cas12a carrying vector,
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<p style="margin-top: 1em;">
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nearly two weeks had passed, indicating that working with this vector can be quite tedious and
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After the successful Gibson assembly of crRNA and repair template into the Cas12a carrying
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time consuming. This is one of the many reasons why we chose to implement such a CRISPR system
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vector, nearly two weeks had passed, indicating that working with this vector can be quite
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into our MoClo based toolbox. While building this system we made sure to directly prove it by
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tedious and time consuming. This is one of the many reasons why we chose to implement such a
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using it to reverse this point mutation, making sure that we tackle this crucial goal through
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CRISPR system into our MoClo based toolbox. While building this system we made sure to directly
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multiple approaches.
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prove it by using it to reverse this point mutation, making sure that we tackle this crucial
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goal through multiple approaches.
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Revision as of 17:56, 18 November 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/Cpf1 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 get more and more relevance 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

We modified Synechococcus elongatus UTEX 2973 to establish the CRISPR/Cpf1 system in our organism.

T O O L B O X

We expanded last years Marburg Collection and made the parts suitable for Synechococcus elongatus UTEX 2973.