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| + | |
| | | |
− | <!--------------------------------------- TWO COLUMN IMG LEFT ------------------------------------------------>
| + | <!--------------------------------------- TWO COLUMN IMG LEFT ------------------------------------------------> |
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− | <div class="two-columns" style="margin-top:0px">
| + | <div class="two-columns" style="margin-top:0px"> |
| | | |
− | <div>
| + | <div> |
− | <!-- IMAGE WITH CAPTION -->
| + | <!-- IMAGE WITH CAPTION --> |
− | <figure class="is-revealing">
| + | <figure class="is-revealing"> |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d9/T--Humboldt_Berlin--designfig2.png" alt="cloning strategy" />
| + | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d9/T--Humboldt_Berlin--designfig2.png" alt="cloning strategy" /> |
− | </figure>
| + | </figure> |
− | </div>
| + | </div> |
− | <div>
| + | <div> |
− | <p><b>Fig. 2. Overview of the Golden Gate cloning strategy.</b>
| + | <p><b>Fig. 2. Overview of the Golden Gate cloning strategy.</b> |
− | <p>
| + | <p> |
− | <figcaption> Multiple basic genetic elements on a level 0 vector (Phytobricks) can be assembled to a full transcription unit on a level 1 vector. Specific fusion sites (shown in grey) and BpiI recognition sites are added to new genetic elements via PCR. To build a L0 construct the Type IIS restriction enzyme BpiI digests the PCR fragment and the L0 vector. The L0 vector, in turn, contains the recognition site for BsaI. Digestion with BsaI and ligation of several L0 parts with a L1 backbone leads to a L1 transcriptional unit. The different L1 modules of choice can then be assembled via BpiI into the final L2 construct in which no recognition sites for type IIS restriction enzymes are left.</figcaption>
| + | <figcaption> Multiple basic genetic elements on a level 0 vector (Phytobricks) can be assembled to a full transcription unit on a level 1 vector. Specific fusion sites (shown in grey) and BpiI recognition sites are added to new genetic elements via PCR. To build a L0 construct the Type IIS restriction enzyme BpiI digests the PCR fragment and the L0 vector. The L0 vector, in turn, contains the recognition site for BsaI. Digestion with BsaI and ligation of several L0 parts with a L1 backbone leads to a L1 transcriptional unit. The different L1 modules of choice can then be assembled via BpiI into the final L2 construct in which no recognition sites for type IIS restriction enzymes are left.</figcaption> |
− | </div>
| + | </div> |
− | </div>
| + | </div> |
| | | |
− | <!--------------------------------------- TWO COLUMN IMG LEFT END ----------------------------------------------->
| + | <!--------------------------------------- TWO COLUMN IMG LEFT END -----------------------------------------------> |
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− | <!------------------------------------------ TWO COLUMN IMG RIGHT ------------------------------------------------>
| + | <!------------------------------------------ TWO COLUMN IMG RIGHT ------------------------------------------------> |
| | | |
− | <div class="two-columns-headline" id="SelectionCassette">
| + | <div class="two-columns-headline" id="SelectionCassette"> |
− | <!----------------------------------------------------------------------->
| + | <!-----------------------------------------------------------------------> |
− | <!------------------Selection Cassette construction----------------------->
| + | <!------------------Selection Cassette construction-----------------------> |
− | <!----------------------------------------------------------------------->
| + | <!-----------------------------------------------------------------------> |
− | <h3 class="headline3">1.2 Construction of a selection cassette</h3>
| + | <h3 class="headline3">1.2 Construction of a selection cassette</h3> |
− | </div>
| + | </div> |
− | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px">
| + | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> |
− | <div>
| + | <div> |
− | <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>.
| + | <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>. |
− | </p>
| + | </p> |
− | </br>
| + | </br> |
− | <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. |
− | </p>
| + | </p> |
− | </div>
| + | </div> |
− | <div>
| + | <div> |
− | <!--- IMAGE --->
| + | <!--- IMAGE ---> |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/b/bc/T--Humboldt_Berlin--Selection_cassette.jpeg" alt="Selection cassette" />
| + | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/b/bc/T--Humboldt_Berlin--Selection_cassette.jpeg" alt="Selection cassette" /> |
− | <p><b>Fig. 3. Cloning process to insert a selection cassette into the L1c-RFP_ampR/Ori plasmid.</b>
| + | <p><b>Fig. 3. Cloning process to insert a selection cassette into the L1c-RFP_ampR/Ori plasmid.</b> |
− | <p>
| + | <p> |
− | <figcaption> The selection transcription unit (PsaD-B2Linker-hygR-Rbcs2) inside the pICH47732 backbone can be amplified with primers containing restriction sites for BamHI and XhoI to receive a PCR product linkable with the new constructed L1 plasmid L1c-RFP_ampR/Ori when digested with BamHI and XhoI. The resulting plasmid L1c-RFP-HygR represents a level 1 Golden Gate cloning vector specified for <i>C. reinhardtii</i>. </figcaption>
| + | <figcaption> The selection transcription unit (PsaD-B2Linker-hygR-Rbcs2) inside the pICH47732 backbone can be amplified with primers containing restriction sites for BamHI and XhoI to receive a PCR product linkable with the new constructed L1 plasmid L1c-RFP_ampR/Ori when digested with BamHI and XhoI. The resulting plasmid L1c-RFP-HygR represents a level 1 Golden Gate cloning vector specified for <i>C. reinhardtii</i>. </figcaption> |
| | | |
− | </div>
| + | </div> |
− | </div>
| + | </div> |
| | | |
− | <!--------------------------------------- TWO COLUMN IMG RIGHT END ------------------------------------------------>
| + | <!--------------------------------------- TWO COLUMN IMG RIGHT END ------------------------------------------------> |
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− | <!--------------------------------------- TWO COLUMN IMG LEFT ------------------------------------------------>
| + | <!--------------------------------------- TWO COLUMN IMG LEFT ------------------------------------------------> |
| | | |
− | <div class="two-columns-headline" id="Expression">
| + | <div class="two-columns-headline" id="Expression"> |
− | <h3 class="headline3">1.3 Expression analysis - testing our transgenic proteins</h3>
| + | <h3 class="headline3">1.3 Expression analysis - testing our transgenic proteins</h3> |
− | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px">
| + | |
− | <!-- IMAGE -->
| + | |
− | <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> |
− | <div> | + | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> |
− | <p> | + | <!-- IMAGE --> |
− | 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. | + | <div> |
− | </p>
| + | <figure class="is-revealing"> |
− | <p>
| + | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d5/T--Humboldt_Berlin--screening.jpeg" /> |
− | 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).
| + | </figure> |
− | </p>
| + | </div> |
− | <p>
| + | <div> |
− | 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> |
− | </p>
| + | 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> |
| + | <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> | | </div> |
− | </div>
| |
| | | |
− | <!--------------------------------------- TWO COLUMN IMG LEFT END ----------------------------------------------->
| + | <!--------------------------------------- TWO COLUMN IMG LEFT END -----------------------------------------------> |
− | <!------------------------------------------ TWO COLUMN IMG RIGHT ------------------------------------------------>
| + | <!------------------------------------------ TWO COLUMN IMG RIGHT ------------------------------------------------> |
| | | |
− | <div class="two-columns-headline" id="Secretion">
| + | <div class="two-columns-headline" id="Secretion"> |
− | <!----------------------------------------------------------------------->
| + | <!-----------------------------------------------------------------------> |
− | <!------------------Secretion signals------------------------------------>
| + | <!------------------Secretion signals------------------------------------> |
− | <!----------------------------------------------------------------------->
| + | <!-----------------------------------------------------------------------> |
− | <h3 class="headline3">3.1 Testing secretion signals</h3>
| + | <h3 class="headline3">3.1 Testing secretion signals</h3> |
− | </div>
| + | |
− | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px">
| + | |
− | <div>
| + | |
− | <p>
| + | |
− | As many enzymes function outside of its host organism, we want to recreate the secretion of heterologous expressed proteins in <i>C. reinhardtii</i>. Secretion is possible due to a signal sequence attached to the protein at its 3’UTR that leads it to the secretory pathway. We take native signals from arylsulphatase 1 (ARS1) and a putative signal sequence of gametolysin (GLE) (Ramos-Martinez, Fimognari, & Sakuragi, 2017; Rasala et al., 2012) and build L0 modules with them. As the yield of heterologous expressed proteins fused to these secretion signals was previously described as relatively low, we additionally designed a 5’UTR glycomodule (SP<sub>20</sub>) as a L0 part which also enhances the stability of the attached protein when secreted (Ramos-Martinez, Fimognari, & Sakuragi, 2017; Rasala et al., 2012).
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | In order to test these secretion signals, we tagged them with a YFP protein, which can be observed under a fluorescence microscope (excitation peak at 480 nm and emission peak at 528 nm). If secreted, the medium outside the cells is expected to emit fluorescence as opposed to the medium surrounding wild type algae.
| + | |
− | </p>
| + | |
| </div> | | </div> |
− | <div> | + | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> |
− | <!--- IMAGE ---> | + | <div> |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/8/89/T--Humboldt_Berlin--Secretion_icon.png" />
| + | <p> |
| + | As many enzymes function outside of its host organism, we want to recreate the secretion of heterologous expressed proteins in <i>C. reinhardtii</i>. Secretion is possible due to a signal sequence attached to the protein at its 3’UTR that leads it to the secretory pathway. We take native signals from arylsulphatase 1 (ARS1) and a putative signal sequence of gametolysin (GLE) (Ramos-Martinez, Fimognari, & Sakuragi, 2017; Rasala et al., 2012) and build L0 modules with them. As the yield of heterologous expressed proteins fused to these secretion signals was previously described as relatively low, we additionally designed a 5’UTR glycomodule (SP<sub>20</sub>) as a L0 part which also enhances the stability of the attached protein when secreted (Ramos-Martinez, Fimognari, & Sakuragi, 2017; Rasala et al., 2012). |
| + | </p> |
| + | <p> |
| + | In order to test these secretion signals, we tagged them with a YFP protein, which can be observed under a fluorescence microscope (excitation peak at 480 nm and emission peak at 528 nm). If secreted, the medium outside the cells is expected to emit fluorescence as opposed to the medium surrounding wild type algae. |
| + | </p> |
| + | </div> |
| + | <div> |
| + | <!--- IMAGE ---> |
| + | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/8/89/T--Humboldt_Berlin--Secretion_icon.png" /> |
| | | |
| + | </div> |
| </div> | | </div> |
− | </div>
| |
| | | |
− | <!--------------------------------------- TWO COLUMN IMG RIGHT END ------------------------------------------------>
| + | <!--------------------------------------- TWO COLUMN IMG RIGHT END ------------------------------------------------> |
− | <!--------------------------------------- TWO COLUMN IMG LEFT ------------------------------------------------>
| + | <!--------------------------------------- TWO COLUMN IMG LEFT ------------------------------------------------> |
| | | |
− | <div class="two-columns-headline" id="PtxD">
| + | <div class="two-columns-headline" id="PtxD"> |
− | <!----------------------------------------------------------------------->
| + | <!-----------------------------------------------------------------------> |
− | <!--------------------PtxD phosphite marker-------------------->
| + | <!--------------------PtxD phosphite marker--------------------> |
− | <!----------------------------------------------------------------------->
| + | <!-----------------------------------------------------------------------> |
− | <h3 class="headline3">1.3.3 PtxD - Phosphite Oxidoreductase</h3>
| + | <h3 class="headline3">1.3.3 PtxD - Phosphite Oxidoreductase</h3> |
− | </div>
| + | |
− | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px">
| + | |
− | <!-- IMAGE WITH CAPTION -->
| + | |
− | <div>
| + | |
− | <figure class="is-revealing">
| + | |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d5/T--Humboldt_Berlin--screening.jpeg" />
| + | |
− | <p><b>Fig. </b>
| + | |
− | <p>
| + | |
− | <figcaption> This figure shows how the phosphite marker works. </figcaption>
| + | |
− | </figure>
| + | |
| </div> | | </div> |
− | <div> | + | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> |
− | <p>
| + | |
− | Algae depend on phosphate as their sole phosphorus source. Phosphite, the reduced form of phosphate however, competes with phosphate for transport proteins and therefore is harmful to plant growth. The phosphite oxidoreductase from <i>Pseudomonas stutzeri WM88</i> (PtxD) oxidizes phosphite to phosphate using NAD<sup>+</sup> as a co-substrate (Loera-Quezada et al., 2016; White & Metcalf, 2007). We provide our <i>C. reinhardtii</i> strains with the PtxD enzyme to increase its competitiveness against other microorganisms in culture. This way, we enable it to degrade phosphite, presumably as only organism in our culture, avoiding contamination.
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | We observe the growth of our engineered <i>C. reinhardtii</i> in media containing phosphite, phosphate or phosphorus to prove the enzyme activity. In the phosphite-supplied media, we expect to observe growth of the strains expressing PtxD and death of wild type algae due to a lack of phosphorus.
| + | |
− | </p>
| + | |
− | </div>
| + | |
− | </div>
| + | |
− | <!--------------------------------------- TWO COLUMN IMG LEFT END ----------------------------------------------->
| + | |
− | | + | |
− | <!------------------------------------------ TWO COLUMN IMG RIGHT ------------------------------------------------>
| + | |
− | | + | |
− | <div class="two-columns-headline" id="Cas9">
| + | |
− | <!----------------------------------------------------------------------->
| + | |
− | <!------------------Cas9 site-directed mutagenesis------------------------------------>
| + | |
− | <!----------------------------------------------------------------------->
| + | |
− | <h3 class="headline3">1.3.4 Cas9/sgRNA-mediated site-directed mutagenesis </h3>
| + | |
− | </div>
| + | |
− | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px">
| + | |
− | <div>
| + | |
− | <p>
| + | |
− | When establishing <i>Chlamydomonas</i> algae as a viable chassis for expression of synthetic constructs, one must consider how to ramp up the expression of such constructs. One solution might be to guide the insertion into a certain genome locus. As has been proven before, gene insertions into the nuclear genome of <i>C. reinhardtii</i> can be achieved by transformation of a recombinant Cas9 protein, combined with a guide RNA (gRNA) (Kelterborn et al., <i>unpublished data</i>).
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | Target recognition inside the nuclear genome of <i>C. reinhardtii</i> relies on the presence of protospacers and protospacer-adjacent motifs (PAM) on the target DNA. A gRNA, which is designed to match specific regions on the genome and bind to them (Mali et al., 2013) is used to direct the Cas9 endonuclease. Upon binding to the target region, a cut of Cas9 creates a double-stranded break (DSB) with blunt ends. Into the DSB artificial DNA could be inserted through DNA repair-mechanisms. We wanted to find out if there were regions in the genome of <i>C. reinhardtii</i> that displayed an increased protein expression. To this end, we designed sgRNA complementary to three different genome regions.
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | The first locus is the SNRK locus. Since Kelterborn et al. had implemented a straight-forward screening process, successful cleavages could easily be identified. We want to use these loci mainly as a comparison to different locus insertions, since we do not expect high expression. As we are using the Promoter of the Photosystem Subunit II, we want to check if we are able to increase the expression by inserting our DNA Fragments at the end of the PsaD gene. Strenkert et al. have shown that a light dependent increase of PsaD expression occurs. When grown under synchronized light conditions a higher protein yield may be achieved.
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | Ribulose-1,5-bisphosphat-carboxylase (RuBisCo) is often referred to as the most abundant soluble protein. Carrying the function of carbon dioxide fixation it is present in all plants and phototrophic organisms. Due to this frequent appearance, we want to see if insertions inside the RuBisCo locus lead to higher expression. By targeting this locus for the insertion of our constructs, we hope to increase the protein yield, while characterizing the expression pattern of <i>C. reinhardtii</i> to ease the further use for the SynBio community.
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | During the process of electroporation transgenic DNA is inserted randomly into the genome of <i>C. reinhardtii</i>. This opens up the possibility that a high expression locus might be found by coincidence during our screening process. This locus could then be considered for further transformations to yield a high protein expression, useful in all genetic engineering projects.
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | To identify such a locus we try to amplify this genomic region with a <b>restriction enzyme site-directed amplification PCR (RESDA-PCR)</b>. This polymerase chain reaction uses primers that are designed in a way that they anneal at restriction enzyme recognition sites of the enzymes <i>Alu</i>I, <i>Sac</i>II, PstI or <i>Taq</i>I. These sites are widely spread inside the genome. This increases the probability that the transgene DNA is inserted next to one of these sites, so that a PCR-product containing the genome sequences might be amplified and sequenced.
| + | |
− | </p>
| + | |
− | </div>
| + | |
− | <div>
| + | |
| <!-- IMAGE WITH CAPTION --> | | <!-- IMAGE WITH CAPTION --> |
| <div> | | <div> |
Line 371: |
Line 320: |
| <p><b>Fig. </b> | | <p><b>Fig. </b> |
| <p> | | <p> |
− | <figcaption> Here should be a figure depicting the Cas9 and/or the RESDA-System. </figcaption> | + | <figcaption> This figure shows how the phosphite marker works. </figcaption> |
| </figure> | | </figure> |
| + | </div> |
| + | <div> |
| + | <p> |
| + | Algae depend on phosphate as their sole phosphorus source. Phosphite, the reduced form of phosphate however, competes with phosphate for transport proteins and therefore is harmful to plant growth. The phosphite oxidoreductase from <i>Pseudomonas stutzeri WM88</i> (PtxD) oxidizes phosphite to phosphate using NAD<sup>+</sup> as a co-substrate (Loera-Quezada et al., 2016; White & Metcalf, 2007). We provide our <i>C. reinhardtii</i> strains with the PtxD enzyme to increase its competitiveness against other microorganisms in culture. This way, we enable it to degrade phosphite, presumably as only organism in our culture, avoiding contamination. |
| + | </p> |
| + | <p> |
| + | We observe the growth of our engineered <i>C. reinhardtii</i> in media containing phosphite, phosphate or phosphorus to prove the enzyme activity. In the phosphite-supplied media, we expect to observe growth of the strains expressing PtxD and death of wild type algae due to a lack of phosphorus. |
| + | </p> |
| </div> | | </div> |
| </div> | | </div> |
− | </div>
| + | <!--------------------------------------- TWO COLUMN IMG LEFT END -----------------------------------------------> |
− | <!--------------------------------------- TWO COLUMN IMG RIGHT END ------------------------------------------------>
| + | |
− | <!--------------------------------------- TWO COLUMN IMG LEFT ------------------------------------------------>
| + | |
| | | |
− | <div class="two-columns-headline" id="GrowthModeling">
| + | <!------------------------------------------ TWO COLUMN IMG RIGHT ------------------------------------------------> |
− | <!-----------------------------------------------------------------------> | + | |
− | <!--------------------Modeling photoautotrophic growth--------------------> | + | <div class="two-columns-headline" id="Cas9"> |
− | <!----------------------------------------------------------------------->
| + | <!-----------------------------------------------------------------------> |
− | <h3 class="headline3">1.3.5. Modeling photoautotrophic growth of Chlamy</h3>
| + | <!------------------Cas9 site-directed mutagenesis------------------------------------> |
| + | <!-----------------------------------------------------------------------> |
| + | <h3 class="headline3">1.3.4 Cas9/sgRNA-mediated site-directed mutagenesis </h3> |
| + | </div> |
| + | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> |
| + | <div> |
| + | <p> |
| + | When establishing <i>Chlamydomonas</i> algae as a viable chassis for expression of synthetic constructs, one must consider how to ramp up the expression of such constructs. One solution might be to guide the insertion into a certain genome locus. As has been proven before, gene insertions into the nuclear genome of <i>C. reinhardtii</i> can be achieved by transformation of a recombinant Cas9 protein, combined with a guide RNA (gRNA) (Kelterborn et al., <i>unpublished data</i>). |
| + | </p> |
| + | <p> |
| + | Target recognition inside the nuclear genome of <i>C. reinhardtii</i> relies on the presence of protospacers and protospacer-adjacent motifs (PAM) on the target DNA. A gRNA, which is designed to match specific regions on the genome and bind to them (Mali et al., 2013) is used to direct the Cas9 endonuclease. Upon binding to the target region, a cut of Cas9 creates a double-stranded break (DSB) with blunt ends. Into the DSB artificial DNA could be inserted through DNA repair-mechanisms. We wanted to find out if there were regions in the genome of <i>C. reinhardtii</i> that displayed an increased protein expression. To this end, we designed sgRNA complementary to three different genome regions. |
| + | </p> |
| + | <p> |
| + | The first locus is the SNRK locus. Since Kelterborn et al. had implemented a straight-forward screening process, successful cleavages could easily be identified. We want to use these loci mainly as a comparison to different locus insertions, since we do not expect high expression. As we are using the Promoter of the Photosystem Subunit II, we want to check if we are able to increase the expression by inserting our DNA Fragments at the end of the PsaD gene. Strenkert et al. have shown that a light dependent increase of PsaD expression occurs. When grown under synchronized light conditions a higher protein yield may be achieved. |
| + | </p> |
| + | <p> |
| + | Ribulose-1,5-bisphosphat-carboxylase (RuBisCo) is often referred to as the most abundant soluble protein. Carrying the function of carbon dioxide fixation it is present in all plants and phototrophic organisms. Due to this frequent appearance, we want to see if insertions inside the RuBisCo locus lead to higher expression. By targeting this locus for the insertion of our constructs, we hope to increase the protein yield, while characterizing the expression pattern of <i>C. reinhardtii</i> to ease the further use for the SynBio community. |
| + | </p> |
| + | <p> |
| + | During the process of electroporation transgenic DNA is inserted randomly into the genome of <i>C. reinhardtii</i>. This opens up the possibility that a high expression locus might be found by coincidence during our screening process. This locus could then be considered for further transformations to yield a high protein expression, useful in all genetic engineering projects. |
| + | </p> |
| + | <p> |
| + | To identify such a locus we try to amplify this genomic region with a <b>restriction enzyme site-directed amplification PCR (RESDA-PCR)</b>. This polymerase chain reaction uses primers that are designed in a way that they anneal at restriction enzyme recognition sites of the enzymes <i>Alu</i>I, <i>Sac</i>II, PstI or <i>Taq</i>I. These sites are widely spread inside the genome. This increases the probability that the transgene DNA is inserted next to one of these sites, so that a PCR-product containing the genome sequences might be amplified and sequenced. |
| + | </p> |
| + | </div> |
| + | <div> |
| + | <!-- IMAGE WITH CAPTION --> |
| + | <div> |
| + | <figure class="is-revealing"> |
| + | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/d/d5/T--Humboldt_Berlin--screening.jpeg" /> |
| + | <p><b>Fig. </b> |
| + | <p> |
| + | <figcaption> Here should be a figure depicting the Cas9 and/or the RESDA-System. </figcaption> |
| + | </figure> |
| + | </div> |
| + | </div> |
| + | </div> |
| + | <!--------------------------------------- TWO COLUMN IMG RIGHT END ------------------------------------------------> |
| + | <!--------------------------------------- TWO COLUMN IMG LEFT ------------------------------------------------> |
| + | |
| + | <div class="two-columns-headline" id="GrowthModeling"> |
| + | <!-----------------------------------------------------------------------> |
| + | <!--------------------Modeling photoautotrophic growth--------------------> |
| + | <!-----------------------------------------------------------------------> |
| + | <h3 class="headline3">1.3.5. Modeling photoautotrophic growth of Chlamy</h3> |
| + | </div> |
| <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> | | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> |
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| <!-------------------Modeling PET degradation----------------------> | | <!-------------------Modeling PET degradation----------------------> |
| <!-----------------------------------------------------------------------> | | <!-----------------------------------------------------------------------> |
| + | <h3 class="headline3" :>2.1 Modeling PET degradation by <i>C. reinhardtii</i> using an optimized PETase</h3> |
| </div> | | </div> |
− | <h3 class="headline3" :>2.1 Modeling PET degradation by <i>C. reinhardtii</i> using an optimized PETase</h3>
| + | |
| <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> | | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> |
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| <div class="two-columns-headline" id="PETModelling"> | | <div class="two-columns-headline" id="PETModelling"> |
− | | + | <h3 class="headline3" :>2.2 Transformation of PETase and MHETase into <i>C. reinhardtii</i></h3> |
| <!-----------------------------------------------------------------------> | | <!-----------------------------------------------------------------------> |
| <!-------------------Transformation of C. reinhardtii----------------------> | | <!-------------------Transformation of C. reinhardtii----------------------> |
| <!-----------------------------------------------------------------------> | | <!-----------------------------------------------------------------------> |
| </div> | | </div> |
− | <h3 class="headline3" :>2.2 Transformation of PETase and MHETase into <i>C. reinhardtii</i></h3>
| + | |
| <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> | | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> |
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| </div> | | </div> |
− | </div>
| |
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| + | <!--------------------------------------- TWO COLUMN IMG RIGHT END ------------------------------------------------> |
| | | |
− | <!------------------------------------------ DEVIDER LINE -------------------------------------------->
| + | <!------------------------------------------ DEVIDER LINE --------------------------------------------> |
− | <div class="greyblue-devider"></div>
| + | <div class="greyblue-devider"></div> |
− | <!------------------------------------------ DEVIDER LINE END -------------------------------------------->
| + | <!------------------------------------------ DEVIDER LINE END --------------------------------------------> |
| | | |
− | <section class="width-limit">
| + | <section class="width-limit"> |
− | <div class="two-columns block-text not-centered small-sized">
| + | <div class="two-columns block-text not-centered small-sized"> |
− | <!----------------------------------------------------------------------->
| + | <!-----------------------------------------------------------------------> |
− | <!-------------------------------- SOURCES ------------------------------>
| + | <!-------------------------------- SOURCES ------------------------------> |
− | <!----------------------------------------------------------------------->
| + | <!-----------------------------------------------------------------------> |
− | <div>
| + | <div> |
− | <p>
| + | <p> |
− | Crozet, P., Navarro, F. J., Willmund, F., Mehrshahi, P., Bakowski, K., Lauersen, K. J., ... Lemaire, S. D. (2018). Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synthetic Biology, 7(9), 2074-2086. Retrieved from https://doi.org/10.1021/acssynbio.8b00251. doi:10.1021/acssynbio.8b00251
| + | Crozet, P., Navarro, F. J., Willmund, F., Mehrshahi, P., Bakowski, K., Lauersen, K. J., ... Lemaire, S. D. (2018). Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synthetic Biology, 7(9), 2074-2086. Retrieved from https://doi.org/10.1021/acssynbio.8b00251. doi:10.1021/acssynbio.8b00251 |
− | </p>
| + | </p> |
− | <p>
| + | <p> |
− | Ebrahim, A., Lerman, J. A., Palsson, B. O., & Hyduke, D. R. (2013). COBRApy: COnstraints-Based Reconstruction and Analysis for Python. BMC Systems Biology, 7(1), 74. https://doi.org/10.1186/1752-0509-7-74
| + | Ebrahim, A., Lerman, J. A., Palsson, B. O., & Hyduke, D. R. (2013). COBRApy: COnstraints-Based Reconstruction and Analysis for Python. BMC Systems Biology, 7(1), 74. https://doi.org/10.1186/1752-0509-7-74 |
− | </p>
| + | </p> |
− | <p>
| + | <p> |
− | Engler, C., Kandzia, R. & Marillonnet, S. (2008). A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLOS ONE, 3(11), e3647. Retrieved from https://doi.org/10.1371/journal.pone.0003647. doi:10.1371/journal.pone.0003647
| + | Engler, C., Kandzia, R. & Marillonnet, S. (2008). A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLOS ONE, 3(11), e3647. Retrieved from https://doi.org/10.1371/journal.pone.0003647. doi:10.1371/journal.pone.0003647 |
− | </p>
| + | </p> |
− | <p>
| + | <p> |
− | Greiner, A., Kelterborn, S., Evers, H., Kreimer, G., Sizova, I. & Hegemann, P. (2017). Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9. The Plant Cell. 29. tpc.00659.2017. 10.1105/tpc.17.00659.
| + | Greiner, A., Kelterborn, S., Evers, H., Kreimer, G., Sizova, I. & Hegemann, P. (2017). Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9. The Plant Cell. 29. tpc.00659.2017. 10.1105/tpc.17.00659. |
− | </p>
| + | </p> |
− | <p>
| + | <p> |
− | Imam, S. , Schäuble, S. , Valenzuela, J. , López García de Lomana, A. , Carter, W. , Price, N. D. and Baliga, N. S. (2015), A refined genome‐scale reconstruction of Chlamydomonas metabolism provides a platform for systems‐level analyses. Plant J, 84: 1239-1256. doi:10.1111/tpj.13059
| + | Imam, S. , Schäuble, S. , Valenzuela, J. , López García de Lomana, A. , Carter, W. , Price, N. D. and Baliga, N. S. (2015), A refined genome‐scale reconstruction of Chlamydomonas metabolism provides a platform for systems‐level analyses. Plant J, 84: 1239-1256. doi:10.1111/tpj.13059 |
− | </p>
| + | </p> |
− | <p>
| + | <p> |
− | Kelterborn, S., Boehning, F., Evers, H., Sizova, I., Baidukova, O., & Hegemann, P. (2019). Gene editing in green alga Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Unpublished work.
| + | Kelterborn, S., Boehning, F., Evers, H., Sizova, I., Baidukova, O., & Hegemann, P. (2019). Gene editing in green alga Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Unpublished work. |
− | </p>
| + | </p> |
− | Kliphuis, A. M. J., Klok, A. J., Martens, D. E., Lamers, P. P., Janssen, M., & Wijffels, R. H. (2012). Metabolic modeling of Chlamydomonas reinhardtii: Energy requirements for photoautotrophic growth and maintenance. Journal of Applied Phycology, 24(2), 253–266. https://doi.org/10.1007/s10811-011-9674-3
| + | Kliphuis, A. M. J., Klok, A. J., Martens, D. E., Lamers, P. P., Janssen, M., & Wijffels, R. H. (2012). Metabolic modeling of Chlamydomonas reinhardtii: Energy requirements for photoautotrophic growth and maintenance. Journal of Applied Phycology, 24(2), 253–266. https://doi.org/10.1007/s10811-011-9674-3 |
− | </p>
| + | </p> |
− | <p>
| + | <p> |
− | Loera‐Quezada, M. M., Leyva‐González, M. A., Velázquez‐Juárez, G. , Sanchez‐Calderón, L. , Do Nascimento, M. , López‐Arredondo, D. and Herrera‐Estrella, L. (2016), A novel genetic engineering platform for the effective management of biological contaminants for the production of microalgae. Plant Biotechnol J, 14: 2066-2076. doi:10.1111/pbi.12564
| + | Loera‐Quezada, M. M., Leyva‐González, M. A., Velázquez‐Juárez, G. , Sanchez‐Calderón, L. , Do Nascimento, M. , López‐Arredondo, D. and Herrera‐Estrella, L. (2016), A novel genetic engineering platform for the effective management of biological contaminants for the production of microalgae. Plant Biotechnol J, 14: 2066-2076. doi:10.1111/pbi.12564 |
− | </p> | + | </p> |
| + | </div> |
| + | <div> |
| + | <p> |
| + | Ma, Y., Yao, M., Li, B., Ding, M., He, B., Chen, S., ... & Yuan, Y. (2018). Enhanced poly (ethylene terephthalate) hydrolase activity by protein engineering. Engineering, 4(6), 888-893. Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., and Church, G. M. “RNA-Guided Human Genome Engineering via Cas9,” Science, vol. 339, pp. 823–826, feb 2013. |
| + | </p> |
| + | <p> |
| + | Merchant, S. S., Prochnik, S. E., Vallon, O., Harris, E. H., Karpowicz, S. J., Witman, G. B., … Grossman, A. R. (2007). The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science (New York, N.Y.), 318(5848), 245–250. doi:10.1126/science.1143609 |
| + | </p> |
| + | <p> |
| + | Patron, N. J., Orzaez, D., Marillonnet, S., Warzecha, H., Matthewman, C., Youles, M., . . . Haseloff, J. (2015). Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist, 208(1), 13-19. Retrieved from https://doi.org/10.1111/nph.13532. doi:10.1111/nph.13532 |
| + | </p> |
| + | <p> |
| + | Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS ONE, 6(2), e16765. Retrieved from https://doi.org/10.1371/journal.pone.0016765. doi:10.1371/journal.pone.0016765 |
| + | </p> |
| + | <p> |
| + | Palm, G. J., Reisky, L., Böttcher, D., Müller, H., Michels, E. A., Walczak, M. C., ... & Weber, G. (2019). Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate. Nature communications, 10(1), 1717. |
| + | </p> |
| + | <p> |
| + | Strenkert, S., Schmollinger, S., Gallaher, S. D., Salomé, P. A., Purvine, S. O., Nicora, C. D., Mettler-Altmann, T., Soubeyrand, E., Weber, A. P. M., Lipton, M. S., Basset, G. J., Merchant, S. S. Proceedings of the National Academy of Sciences Feb 2019, 116 (6) 2374-2383; DOI:10.1073/pnas.1815238116 |
| + | </p> |
| + | <p> |
| + | </div> |
| </div> | | </div> |
− | <div>
| + | </section> |
− | <p>
| + | |
− | Ma, Y., Yao, M., Li, B., Ding, M., He, B., Chen, S., ... & Yuan, Y. (2018). Enhanced poly (ethylene terephthalate) hydrolase activity by protein engineering. Engineering, 4(6), 888-893. Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., and Church, G. M. “RNA-Guided Human Genome Engineering via Cas9,” Science, vol. 339, pp. 823–826, feb 2013.
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | Merchant, S. S., Prochnik, S. E., Vallon, O., Harris, E. H., Karpowicz, S. J., Witman, G. B., … Grossman, A. R. (2007). The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science (New York, N.Y.), 318(5848), 245–250. doi:10.1126/science.1143609
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | Patron, N. J., Orzaez, D., Marillonnet, S., Warzecha, H., Matthewman, C., Youles, M., . . . Haseloff, J. (2015). Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist, 208(1), 13-19. Retrieved from https://doi.org/10.1111/nph.13532. doi:10.1111/nph.13532
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS ONE, 6(2), e16765. Retrieved from https://doi.org/10.1371/journal.pone.0016765. doi:10.1371/journal.pone.0016765
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | Palm, G. J., Reisky, L., Böttcher, D., Müller, H., Michels, E. A., Walczak, M. C., ... & Weber, G. (2019). Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate. Nature communications, 10(1), 1717.
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | Strenkert, S., Schmollinger, S., Gallaher, S. D., Salomé, P. A., Purvine, S. O., Nicora, C. D., Mettler-Altmann, T., Soubeyrand, E., Weber, A. P. M., Lipton, M. S., Basset, G. J., Merchant, S. S. Proceedings of the National Academy of Sciences Feb 2019, 116 (6) 2374-2383; DOI:10.1073/pnas.1815238116
| + | |
− | </p>
| + | |
− | <p>
| + | |
− | </div>
| + | |
− | </div> | + | |
− | </section>
| + | |
| </section> | | </section> |
| | | |