Difference between revisions of "Team:Humboldt Berlin/Demonstrate"

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<h3>★  ALERT! </h3>
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<p>This page is used by the judges to evaluate your team for the <a href="https://2019.igem.org/Judging/Medals">medal criterion</a> or <a href="https://2019.igem.org/Judging/Awards"> award listed below</a>. </p>
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<p> Delete this box in order to be evaluated for this medal criterion and/or award. See more information at <a href="https://2019.igem.org/Judging/Pages_for_Awards"> Instructions for Pages for awards</a>.</p>
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            </section>
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            <h1 class="page-headline description" style="font-size: 12vw;line-height: 8vw;">Demonstrate</h1>
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            <h2 class="page-subheadline">Proof of functionality</h2>
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                        <img src="https://static.igem.org/mediawiki/2019/3/3a/T--Humboldt_Berlin--notebook_s_1203.png" alt="L0-RFP-clone" width="500" />
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                        <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>
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                        <img src="https://static.igem.org/mediawiki/2019/0/0d/T--Humboldt_Berlin--L0-RFP_AR.jpeg" alt="L0-RFP_AR" width="500" />
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                        <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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                        <!----------------------------------------------------------------------->
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                        <!----------------------------BACKBONES---------------------------->
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                        <h3 class="headline3">Synthesized MoClo backbones</h3>
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                        <p class="block-text medium-sized">
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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>
  
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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>
  
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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>
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<h1>Demonstrate</h1>
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<h3>Gold Medal Criterion #4</h3>
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Teams that can show their system working under real world conditions are usually good at impressing the judges in iGEM. To achieve gold medal criterion #4, convince the judges that your engineered system works.
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Your engineered system has to work under realistic conditions. Your system must comply with all <a href="https://2019.igem.org/Safety/Rules">rules</a> and <a href="https://2019.igem.org/Safety/Policies">policies</a> approved by the iGEM Safety Committee. Your system can derive from or make functional a previous iGEM project by your team or by another team. For multi-component projects, the judges may consider the function of individual components.
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To be eligible for this award, you must add clear documentation to this page and delete the alert box at the top of this page.
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                          Furthermore, we designed a L1-backbone (Part <a href="http://parts.igem.org/Part:BBa_K2984002">BBa_K2984002</a>) on which to assemble the transcriptional units for transformation into <i>C. reinhardtii</i>. It contains RFP, a resistance gene against ampicillin and an Ori for <i>E. coli</i> and is flanked by <i>Bsa</i>I restriction sites. L1-constructs can be transformed into and be expressed by <i>C. reinhardtii</i>. Thus it is proven, that the L1 backbone works properly.
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                        <img src="https://static.igem.org/mediawiki/2019/9/9e/T--Humboldt_Berlin--L1c-1.tiff" alt="L1c-construct1" />
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<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>
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Please see the <a href="https://2019.igem.org/Judging/Medals">2019 Medals Page</a> for more information.
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                    <img src="https://static.igem.org/mediawiki/2019/5/50/T--Humboldt_Berlin--YFP_fluorescence_difference_spectrum.png" alt="fluorescence difference spectrum" />
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                        <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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                        <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/4/44/T--Humboldt_Berlin--YFP_klon.tif" alt="fluorescent YFP clone" width="500" />
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                        <figcaption>Fig. 5 - Visualization of fluorescent YFP-expressing clones of <i>C. reinhardtii</i> by a fluorescence microscope at a magnification of 200x. </figcaption>
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                        <!----------------------------------------------------------------------->
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                        <!----------------------------YFP-FLUORESCENCE---------------------------->
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                        <h3 class="headline3">Fluorescence of YFP</h3>
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                        <p class="block-text medium-sized">
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                            The yellow fluorescent protein YFP was used as a fluorescent tag in some of our constructs. The goal of using YFP as a tag was the measurement of enzyme expression and secretion and to screen for successful mutants using YFP as a marker. Additionally, we wanted to use a YFP-expressing <i>C. reinhardtii</i> to analyse possible locus effects on expression. We were able to successfully transform a YFP-expressing <i>C. reinhardtii</i> with a construct of our own design (Part <a href="http://parts.igem.org/Part:BBa_K2984019">BBa_K2984019</a>).</br></br>
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We were able to successfully measure YFP fluorescence intensity and fluorescence spectra of YFP-expressing <i>C. reinhardtii</i> clones in comparison to the wild type (WT), although along the way we faced problems due to the autofluorescence by photosynthetic compartments. The results showed that our clone exhibited a higher fluorescence intensity at 528 nm than the WT (YFP emission peak) and the fluorescence spectrum of YFP confirmed the presence of the yellow fluorescent protein. This YFP-expressing clone also allowed us to characterize the light induction of the PsaD promoter by doing a time-resolved measurement of the fluorescence intensity, which is further characterized on the page of our <a href="http://parts.igem.org/Part:BBa_K2984019">YFP mVenus construct</a> in the iGEM Registry. By demonstrating the light-induction of the PsaD promoter after expression through our L1-backbone vector, we have provided evidence for the functionality of our components. </br></br>
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                        <!------------------PARO-RESISTANCE----------------------->
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                        <h3 class="headline3">Codon-optimized paromomycin resistance gene </h3>
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                        <p class="block-text medium-sized">
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                          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>
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                        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.                                                                                                                                                                       
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By counting the amount of colonies on the plates, we could determine which construct worked best. </br></br>
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                      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.
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<figcaption>Fig. 6 - Total amount of colonies counted for the standard (blue) and improved (orange) resistance.</figcaption>
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                      <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>
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                        <!----------------------------------------------------------------------->
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                        <!----------------------------CHLAMY AS CHASSIS---------------------------->
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                        <!----------------------------------------------------------------------->
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                        <h3 class="headline3">Demonstration of <i> C. reinhardtii </i> as the perfect organism using growth experiments</h3>
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                        <p class="block-text medium-sized">
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                          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>
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Latest revision as of 21:31, 21 October 2019

falcons

Demonstrate

Proof of functionality

L0-RFP-clone
Fig. 1 - Our self-constructed L0-RFP backbone transformed into E. coli (strain DH10B). Through red-/white-selection successful clones were picked and streaked out on LB-agar plates.
L0-RFP_AR
Fig. 2 - AR promoter in L0-RFP-backbones expressed by E. coli (strain DH10B). The white clones represent positive transformants.

Synthesized MoClo backbones

To synthesize the genetic elements for the Chlamy-HUB Collection, we based our design on the Modular Cloning (MoClo) toolkit optimized for C. reinhardtii (Crozet et al., 2018), which follows the Golden Gate cloning method. To read more about the background on the cloning method please visit our Design page .

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.

The L0-RFP (Part BBa_K2984010) contains an origin for bacterial replication (Ori), a resistance gene against spectinomycin for expression by E. coli and the red fluorescent protein RFP for red-/white-selection. It is flanked by restriction sites for BpiI. Parts with matching L0-overhangs according to the MoClo syntax can be inserted into the backbone using BpiI. Successful transformants are white colored. The established backbone worked as intended, demonstrated by successful transformations of all L0-Parts we have designed.

Furthermore, we designed a L1-backbone (Part BBa_K2984002) on which to assemble the transcriptional units for transformation into C. reinhardtii. It contains RFP, a resistance gene against ampicillin and an Ori for E. coli and is flanked by BsaI restriction sites. L1-constructs can be transformed into and be expressed by C. reinhardtii. Thus it is proven, that the L1 backbone works properly.

L1c-construct1
Fig. 3 - This PCR of C. reinhardtii 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.
fluorescence difference spectrum
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
fluorescent YFP clone
Fig. 5 - Visualization of fluorescent YFP-expressing clones of C. reinhardtii by a fluorescence microscope at a magnification of 200x.

Fluorescence of YFP

The yellow fluorescent protein YFP was used as a fluorescent tag in some of our constructs. The goal of using YFP as a tag was the measurement of enzyme expression and secretion and to screen for successful mutants using YFP as a marker. Additionally, we wanted to use a YFP-expressing C. reinhardtii to analyse possible locus effects on expression. We were able to successfully transform a YFP-expressing C. reinhardtii with a construct of our own design (Part BBa_K2984019).

We were able to successfully measure YFP fluorescence intensity and fluorescence spectra of YFP-expressing C. reinhardtii clones in comparison to the wild type (WT), although along the way we faced problems due to the autofluorescence by photosynthetic compartments. The results showed that our clone exhibited a higher fluorescence intensity at 528 nm than the WT (YFP emission peak) and the fluorescence spectrum of YFP confirmed the presence of the yellow fluorescent protein. This YFP-expressing clone also allowed us to characterize the light induction of the PsaD promoter by doing a time-resolved measurement of the fluorescence intensity, which is further characterized on the page of our YFP mVenus construct in the iGEM Registry. By demonstrating the light-induction of the PsaD promoter after expression through our L1-backbone vector, we have provided evidence for the functionality of our components.

Codon-optimized paromomycin resistance gene

Paromomycin is an antibiotic commonly used as a selection marker for engineered C. reinhardtii. To lend the algae resistance against paromomycin we codon-optimized an existing part (Part BBa_K2984006) for use in C. reinhardtii. Our goal was to increase the expression of a phosphotransferase responsible for antibiotic resistance in the transformed clones.

To see if the expression of the aphVIII gene encoding for the aminoglycoside 3’-phosphotransferase was increased, we performed electroporations to transform C. reinhardtii 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.

We counted the total number of C. reinhardtii 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 BBa_K2984006 in the iGEM Registry.

colonies_total
Fig. 6 - Total amount of colonies counted for the standard (blue) and improved (orange) resistance.
AFM Göttingen
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.

Demonstration of C. reinhardtii as the perfect organism using growth experiments

Using a multicultivator we run different growth experiments with C. reinhardtii. 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 C. reinhardtii and does not affect its replication rate. Thus, Chlamy represents an appropriate model organism to tolerate the process of PET degradation.

The biggest advantage of C. reinhardtii 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 Chlamydomonas 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.

Further we demonstrated that phosphate limitation is harmful for C. reinhardtii 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 Results page.

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