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− | Type IIS restriction enzymes cut outside their recognition sites, making them useful in this cloning method for consecutive assembly of fragments. Through the restriction, overhangs are formed which allow the fusion of said genetic fragments to complementary overhangs of the syntax and thereby determine the order of each in a transcriptional unit (Figure 1). These fusion sites allow for the assembly of several fragments in the right order in just one cloning reaction. The used MoClo-kit offers ten different options for the positioning inside a L1 plasmid which are defined by the parts’ functions. | + | Pierre Crozet and his team created a MoClo toolkit adapted for the model organism C. reinhardtii with 119 openly distributed genetic parts (Crozet et al., 2018) including resistance cassettes. However, these resistance cassettes are not located on any L1 plasmid backbone. Consequently, an additional cloning step is required to assemble a level 2 device, since a single level 1 module cannot be transformed without a resistance cassette. To simplify this cloning process, we design a <i>C. reinhardtii</i> specific resistance cassette to serve as a plasmid component for the transformation into <i>Chlamydomonas</i>. |
| </p> | | </p> |
− | <br />
| + | </br> |
− | <img src="https://static.igem.org/mediawiki/2019/1/1b/T--Humboldt_Berlin--designfig1.png" alt="Overview of the hierarchical and modular cloning system" />
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− | <p style="font-size:12px">
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− | <b>Fig. 1. Universal MoClo fusion sites. </b>
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− | <figcaption> 12 fusion sites (Patron, 2015) for the seamless fusion of different level 0 parts. In general, the fusion sites are grouped into 5’ untranslated regions including promoter sequences (grey), the translated coding sequence CDS (blue) followed by 3’ UTRs ending with a terminator (orange). Within these types, various parts (e.g. three different coding sequences) can be designed and combined into one transcription unit when cloned together into a L1 vector. The bold ATG within the B3 fusion site sets the transcription start.</figcaption>
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− | </div>
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− | <div>
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− | <p>
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− | Within the MoClo syntax, there are three different cloning vectors, level 0, 1 and 2 (referred to as “L0”, “L1” and “L2”, respectively). L0 vectors carry one basic genetic fragment or part, L1 vectors are assembled fragments creating a transcriptional unit and L2 are multigenic constructs. Construction of an L0-part is done by flanking a gene of interest with the specific fusion site and the recognition site of BpiI by a PCR reaction. Upon digestion by BpiI it can be inserted into a previously digested L0-backbone. To then clone it into a L1-backbone, it is digested by BsaI, revealing the fusion sites for its assembly in a transcription unit. Lastly, a fusion of several transcription units (L1) into a L2 multigenic device is possible with the MoClo syntax.
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− | </p>
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− | <p>
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− | As part of our contribution to a toolkit usable by future iGEM teams we design and construct not only the parts we intend to use on our goal of PET-degradation but several more, a L0-backbone and L1-backbone.
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− | </p>
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− | <p>
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− | To ensure that all parts were designed correctly we cloned the PCR fragments into a L0 vector. To this end, we used a self-modified version of a L0 backbone containing RFP. After ligation, we transformed the L0 plasmids containing the parts into Escherichia coli and checked for white transformants. Only genes with the correct fusion and restriction sites could be inserted into the L0 backbone resulting in growth of white colonies, since the RFP gene was interrupted. For further verification, we checked the parts by DNA sequencing. We used the same control mechanisms for L1 assembly constructs.
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− | </p>
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− | </div>
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− | </div>
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− | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px">
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− | <div>
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− | <p> Standard vectors for MoClo cloning are equipped with antibiotic resistance cassettes for cloning in <i>E. coli</i>. But since this work is focused on the expression of PET degrading enzymes by <i>C. reinhardtii</i>, the final transformable devices need to provide a selection advantage for these algae. Pierre Crozet and his team created a MoClo toolkit adapted for the model organism C. reinhardtii with 119 openly distributed genetic parts (Crozet et al., 2018) including resistance cassettes. However, these resistance cassettes are not located on any L1 plasmid backbone. Consequently, an additional cloning step is required to assemble a level 2 device, since a single level 1 module cannot be transformed without a resistance cassette. To simplify this cloning process, we design a <i>C. reinhardtii</i> specific resistance cassette to serve as a plasmid component for the transformation into <i>Chlamydomonas</i>.
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− | </p>
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− | </br>
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| <p> | | <p> |
| To build a selection cassette using the Golden Gate cloning standard, each part required for a selection cassette (promoter, linker, marker and terminator) must be cloned into a level 0 backbone. The “marker” can be any antibiotic resistance for C. reinhardtii. Golden Gate cloning using BsaI can assemble the parts into a level 1 backbone which results in an antibiotic resistance transcriptional unit. Using primers amplifying the selection cassette on a L1 MoClo backbone attaching the restriction sites compared to restriction sites on the “new” L1a,b,c vectors. By attaching BamHI and XhoI sites a PCR product arises which is linkable with the new constructed L1 plasmids when digested with BamHI and XhoI. The resulting plasmids can then represent level 1 Golden Gate cloning vectors specified for C. reinhardtii. The picture shows an example to produce a selection cassette containing a hygromycin resistance linkable to a new L1c plasmid. | | To build a selection cassette using the Golden Gate cloning standard, each part required for a selection cassette (promoter, linker, marker and terminator) must be cloned into a level 0 backbone. The “marker” can be any antibiotic resistance for C. reinhardtii. Golden Gate cloning using BsaI can assemble the parts into a level 1 backbone which results in an antibiotic resistance transcriptional unit. Using primers amplifying the selection cassette on a L1 MoClo backbone attaching the restriction sites compared to restriction sites on the “new” L1a,b,c vectors. By attaching BamHI and XhoI sites a PCR product arises which is linkable with the new constructed L1 plasmids when digested with BamHI and XhoI. The resulting plasmids can then represent level 1 Golden Gate cloning vectors specified for C. reinhardtii. The picture shows an example to produce a selection cassette containing a hygromycin resistance linkable to a new L1c plasmid. |
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| + | </div> |
| + | <div class="expandable-readall"> |
| + | <span class="readmore">Read more</span> |
| + | <span class="readless">Read less</span> |
| + | <img src="https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg"> |
| + | </div> |
| + | </div> |
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| + | <div class="two-columns-headline" id="Secretion"> |
| + | <!-----------------------------------------------------------------------> |
| + | <!------------------Expression Analysis------------------------------------> |
| + | <!-----------------------------------------------------------------------> |
| + | <h3 class="headline3">1.3 Expression analysis - testing our transgenic proteins</h3> |
| + | </div> |
| + | |
| + | <div class="expandable-container"> |
| + | <div class="expandable-preview"> |
| + | <p class="medium-sized"> |
| + | The composite parts we created contained different markers, antibiotic resistances or the phosphite marker PtxD, that allow for selection of transformed algae clones. Depending on their intended utility for <i>C. reinhardtii</i>, each of our constructs is screened in a different way to prove their intended function. |
| + | </p> |
| + | <img src="https://static.igem.org/mediawiki/2019/1/1b/T--Humboldt_Berlin--designfig1.png" alt="Overview of the hierarchical and modular cloning system" /> |
| + | </div> |
| + | <div class="expandable-more"> |
| + | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> |
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| + | div> |
| + | <figure class="is-revealing"> |
| + | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d5/T--Humboldt_Berlin--screening.jpeg" /> |
| + | </figure> |
| + | </div> |
| + | |
| + | <div> |
| + | <p> |
| + | Once constructed, the designed plasmids are transformed into the SAG32-11b and UVM4 strains of <i>C. reinhardtii</i> and tested for expression. As a transformation method we chose electroporation, during which two different electric fields are applied to the algal cell. The first, a high voltage pulse applied for relatively short time causes the membrane to form pores and the second, of a low voltage for relatively long time transfers the DNA into the cell. We co-transform each of our constructs containing the transcriptional unit (level 1 constructs) with a plasmid conferring antibiotic resistance (paromomycin or hygromycin, in our case). |
| + | </p> |
| + | <p> |
| + | After transformation, the algae has to be tested for successful uptake of the plasmids. If they are able to grow on TAP-agar plates containing antibiotics, the resistance plasmid was transformed and is proven to work accordingly. Subsequently, the clones have to be screened for the presence of the genetic constructs. A colony PCR is used to amplify the constructs. If the respective bands show up, the clones are continuously cultivated under thriving expression. |
| + | </p> |
| + | </div> |
| + | </div> |
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| <div class="two-columns-headline" id="Expression"> | | <div class="two-columns-headline" id="Expression"> |