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| <section class="fixed-image-header"> | | <section class="fixed-image-header"> |
| <!-- HEADER IMAGE --> | | <!-- HEADER IMAGE --> |
− | <img src="https://static.igem.org/mediawiki/2019/e/e9/T--Humboldt_Berlin--description-header.jpg" alt="notebook" /> | + | <img src="https://static.igem.org/mediawiki/2019/3/3c/T--Humboldt_Berlin--Demonstrate_header.jpg" alt="falcons" /> |
| </section> | | </section> |
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| <section class="page-content fixed-header-content"> | | <section class="page-content fixed-header-content"> |
| <!---------- SUB HEADLINE -------------> | | <!---------- SUB HEADLINE -------------> |
| + | <div class="two-columns" style="margin:0 2vw 20px;margin-bottom:0px"> |
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| <h2 class="page-subheadline">Proof of functionality</h2> | | <h2 class="page-subheadline">Proof of functionality</h2> |
− | | + | </div> |
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| + | <img src="https://static.igem.org/mediawiki/2019/2/23/T--Humboldt_Berlin--functionality.png" style="float:right"/> |
| + | </div> |
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| <div class="width-limit"> | | <div class="width-limit"> |
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| <!-- IMAGE --> | | <!-- IMAGE --> |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/3/3a/T--Humboldt_Berlin--notebook_s_1203.png" alt="L0-RFP-clone" width="500" /> | + | <img src="https://static.igem.org/mediawiki/2019/3/3a/T--Humboldt_Berlin--notebook_s_1203.png" alt="L0-RFP-clone" width="500" /> |
| <figcaption>Fig. 1 - Our self-constructed L0-RFP backbone transformed into <i>E. coli</i> (strain DH10B). Through red-/white-selection successful clones were picked and streaked out on LB-agar plates.</figcaption> | | <figcaption>Fig. 1 - Our self-constructed L0-RFP backbone transformed into <i>E. coli</i> (strain DH10B). Through red-/white-selection successful clones were picked and streaked out on LB-agar plates.</figcaption> |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/0/0d/T--Humboldt_Berlin--L0-RFP_AR.jpeg" alt="L0-RFP_AR" width="500" /> | + | <img src="https://static.igem.org/mediawiki/2019/0/0d/T--Humboldt_Berlin--L0-RFP_AR.jpeg" alt="L0-RFP_AR" width="500" /> |
| <figcaption>Fig. 2 - AR promoter in L0-RFP-backbones expressed by <i>E. coli</i> (strain DH10B). The white clones represent positive transformants.</figcaption> | | <figcaption>Fig. 2 - AR promoter in L0-RFP-backbones expressed by <i>E. coli</i> (strain DH10B). The white clones represent positive transformants.</figcaption> |
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| To synthesize the genetic elements for the Chlamy-HUB Collection, we based our design on the Modular Cloning (MoClo) toolkit optimized for <i>C. reinhardtii</i> (Crozet et al., 2018), which follows the Golden Gate cloning method. To read more about the background on the cloning method please visit our <a href="https://2019.igem.org/Team:Humboldt_Berlin/Design#GoldenGate">Design page </a>.</br></br> | | To synthesize the genetic elements for the Chlamy-HUB Collection, we based our design on the Modular Cloning (MoClo) toolkit optimized for <i>C. reinhardtii</i> (Crozet et al., 2018), which follows the Golden Gate cloning method. To read more about the background on the cloning method please visit our <a href="https://2019.igem.org/Team:Humboldt_Berlin/Design#GoldenGate">Design page </a>.</br></br> |
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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. As part of our contribution to the iGEM Registry, the ChlamyHUB Collection, we registered two cloning vectors. The backbones to these vectors, L0-RFP- and L1-backbones were created by ourselves.</br></br> | + | 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. As part of our contribution to the iGEM Registry, the ChlamyHUB Collection, we registered two cloning vectors. The backbones to these vectors, L0-RFP- and L1-backbones were created by ourselves.</br></br> |
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| The L0-RFP (Part <a href="http://parts.igem.org/Part:BBa_K2984010">BBa_K2984010</a>) contains an origin for bacterial replication (Ori), a resistance gene against spectinomycin for expression by <i>E. coli</i> and the red fluorescent protein RFP for red-/white-selection. It is flanked by restriction sites for <i>Bpi</i>I. Parts with matching L0-overhangs according to the MoClo syntax can be inserted into the backbone using <i>Bpi</i>I. Successful transformants are white colored. The established backbone worked as intended, demonstrated by successful transformations of all L0-Parts we have designed. </br></br> | | The L0-RFP (Part <a href="http://parts.igem.org/Part:BBa_K2984010">BBa_K2984010</a>) contains an origin for bacterial replication (Ori), a resistance gene against spectinomycin for expression by <i>E. coli</i> and the red fluorescent protein RFP for red-/white-selection. It is flanked by restriction sites for <i>Bpi</i>I. Parts with matching L0-overhangs according to the MoClo syntax can be inserted into the backbone using <i>Bpi</i>I. Successful transformants are white colored. The established backbone worked as intended, demonstrated by successful transformations of all L0-Parts we have designed. </br></br> |
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| </p> | | </p> |
| </div> | | </div> |
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| <!--- IMAGE ---> | | <!--- IMAGE ---> |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/9/9e/T--Humboldt_Berlin--L1c-1.tiff" alt="L1c-construct1" /> | + | <img src="https://static.igem.org/mediawiki/2019/9/9e/T--Humboldt_Berlin--L1c-1.tiff" alt="L1c-construct1" /> |
| <figcaption>Fig. 3 - This PCR of <i>C. reinhardtii</i> clones that could grow on selective media shows bands at a length of 1,6 kb for successfully transformed clones. These were further verified by sequencing.</figcaption> | | <figcaption>Fig. 3 - This PCR of <i>C. reinhardtii</i> clones that could grow on selective media shows bands at a length of 1,6 kb for successfully transformed clones. These were further verified by sequencing.</figcaption> |
| </div> | | </div> |
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| <div class="width-limit"> | | <div class="width-limit"> |
| <div class="two-columns"> | | <div class="two-columns"> |
− | <div> | + | <div class="is-revealing"> |
| <!-- IMAGE --> | | <!-- IMAGE --> |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/5/50/T--Humboldt_Berlin--YFP_fluorescence_difference_spectrum.png" alt="fluorescence difference spectrum" /> | + | <img src="https://static.igem.org/mediawiki/2019/5/50/T--Humboldt_Berlin--YFP_fluorescence_difference_spectrum.png" alt="fluorescence difference spectrum" /> |
| <figcaption>Fig. 4 - YFP emission spectrum of a C. reinhardtii clone with YFP with an emission maximum at approximately 530 nm. Difference spectrum of WT and YFP spectra</figcaption> | | <figcaption>Fig. 4 - YFP emission spectrum of a C. reinhardtii clone with YFP with an emission maximum at approximately 530 nm. Difference spectrum of WT and YFP spectra</figcaption> |
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| <h3 class="headline3">Codon-optimized paromomycin resistance gene </h3> | | <h3 class="headline3">Codon-optimized paromomycin resistance gene </h3> |
| <p class="block-text medium-sized"> | | <p class="block-text medium-sized"> |
− | Paromomycin is an antibiotic commonly used as a selection marker for engineered <i>C. reinhardtii</i>. To lend the algae resistance against paromomycin we codon-optimized an existing part (http://parts.igem.org/Part:BBa_K2984006) for use in <i>C. reinhardtii</i>. Our goal was to increase the expression of a phosphotransferase responsible for antibiotic resistance in the transformed clones. <\br><\br> | + | Paromomycin is an antibiotic commonly used as a selection marker for engineered <i>C. reinhardtii</i>. To lend the algae resistance against paromomycin we codon-optimized an existing part (Part <a href="http://parts.igem.org/Part:BBa_K2984006">BBa_K2984006</a>) for use in <i>C. reinhardtii</i>. Our goal was to increase the expression of a phosphotransferase responsible for antibiotic resistance in the transformed clones. </br></br> |
| To see if the expression of the aphVIII gene encoding for the aminoglycoside 3’-phosphotransferase was increased, we performed electroporations to transform <i>C. reinhardtii</i> with the improved paromomycin resistance. We transformed the two paromomycin constructs with standard and improved codon usage. After resuspension and one day recovery in TAP medium, all samples were plated on TAP-agar plates containing a paromomycin concentration of 10 µM. After two weeks of growth, colonies corresponding to each sample were counted. Each colony of C.reinhardtii represents a successful transformation of the resistance and indicates the expression of the aminoglycoside 3’-phosphotransferase. | | To see if the expression of the aphVIII gene encoding for the aminoglycoside 3’-phosphotransferase was increased, we performed electroporations to transform <i>C. reinhardtii</i> with the improved paromomycin resistance. We transformed the two paromomycin constructs with standard and improved codon usage. After resuspension and one day recovery in TAP medium, all samples were plated on TAP-agar plates containing a paromomycin concentration of 10 µM. After two weeks of growth, colonies corresponding to each sample were counted. Each colony of C.reinhardtii represents a successful transformation of the resistance and indicates the expression of the aminoglycoside 3’-phosphotransferase. |
− | By counting the amount of colonies on the plates, we could determine which construct worked best. <\br><\br> | + | By counting the amount of colonies on the plates, we could determine which construct worked best. </br></br> |
| We counted the total number of <i>C. reinhardtii</i> colonies that were transformed with the standard and improved resistance. The results of the total colony count can be seen in Fig. 7. We discovered that the total number of colonies was much higher for the improved plasmid version. The colonies of the samples using the standard usage resulted in a total amount of 175 whereas the improved plasmid version produced 665 colonies. This result indicates that the improved paromomycin resistance works better than the one with standard codon usage. For further characterization on the improved version of the aphVIII gene and its expression efficiency please refer to part <a href="http://parts.igem.org/Part:BBa_K2984006">BBa_K2984006</a> in the iGEM Registry. | | We counted the total number of <i>C. reinhardtii</i> colonies that were transformed with the standard and improved resistance. The results of the total colony count can be seen in Fig. 7. We discovered that the total number of colonies was much higher for the improved plasmid version. The colonies of the samples using the standard usage resulted in a total amount of 175 whereas the improved plasmid version produced 665 colonies. This result indicates that the improved paromomycin resistance works better than the one with standard codon usage. For further characterization on the improved version of the aphVIII gene and its expression efficiency please refer to part <a href="http://parts.igem.org/Part:BBa_K2984006">BBa_K2984006</a> in the iGEM Registry. |
| </p> | | </p> |
| </div> | | </div> |
− | <div> | + | <div class="is-revealing" > |
| <!--- IMAGE ---> | | <!--- IMAGE ---> |
− | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/a/a7/T--Humboldt_Berlin--Colony_Amount_Total.png" alt="colonies_total" > | + | <img src="https://static.igem.org/mediawiki/2019/a/a7/T--Humboldt_Berlin--Colony_Amount_Total.png" alt="colonies_total" > |
| <figcaption>Fig. 6 - Total amount of colonies counted for the standard (blue) and improved (orange) resistance.</figcaption> | | <figcaption>Fig. 6 - Total amount of colonies counted for the standard (blue) and improved (orange) resistance.</figcaption> |
| </div> | | </div> |
| </div> | | </div> |
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| <!--------------------------------------- TWO COLUMN IMG RIGHT END ------------------------------------------------> | | <!--------------------------------------- TWO COLUMN IMG RIGHT END ------------------------------------------------> |
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| + | <img src="https://static.igem.org/mediawiki/2019/7/7d/T--Humboldt_Berlin--goettingen-afm.jpeg" alt="AFM Göttingen" > |
| + | <figcaption>Adhesion forces of Chlamydomonas flagellar on PET, Atomic Force Microscopy. The algae is pushed against the PET surface and pulled off again by the micropipette. During this process the pipette is deflected. Using the deflection and the spring constant of the pipette the adhesion force can be calculated. </figcaption> |
| + | </div> |
| + | <div> |
| + | <!-----------------------------------------------------------------------> |
| + | <!----------------------------CHLAMY AS CHASSIS----------------------------> |
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| + | <h3 class="headline3">Demonstration of <i> C. reinhardtii </i> as the perfect organism using growth experiments</h3> |
| + | <p class="block-text medium-sized"> |
| + | Using a multicultivator we run different growth experiments with <i>C. reinhardtii</i>. By cultivating Chlamy with the degradation products of MHET, ethylene glycol (EG) and terephthalic acid (TPA), we showed that the alga is able to grow under realistically emerging concentrations of these products. We also could demonstrate that microplastic itself is not harmful for <i> C. reinhardtii </i> and does not affect its replication rate. Thus, Chlamy represents an appropriate model organism to tolerate the process of PET degradation. </br></br> |
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| + | The biggest advantage of <i>C. reinhardtii</i> for PET degradation was demonstrated in cooperation with the Max Planck Institute for Dynamics and Self-Organization Göttingen where we tested the adhesion forces of the <i>Chlamydomonas</i> strain SAG 11-32b on PET. We discovered that the strain bound to PET with forces up to 5 nN. The adhesion could be deactivated by incubation under red light. With this photo-switch-ability we hope to trigger the adhesion in our favour.</br></br> |
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| + | Further we demonstrated that phosphate limitation is harmful for <i>C. reinhardtii</i> when its phosphate storage is depleted and that supplementation of media with phosphite inhibits the phosphate metabolism. With this knowledge, the control of phosphorus availability and metabolization presents a suitable way to control growth, selection advantages and co-controlling of genes coupled to selection markers (like PtxD). For more details visit our <a href="/Team:Humboldt_Berlin/Results"> Results page. </a> |
| + | </br></br> |
| + | </p> |
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