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<h4>Achievements:</h4> | <h4>Achievements:</h4> | ||
− | <p>(I) The SpyTag-SpyCatcher system, originally developed by team | + | <p>(I) The SpyTag-SpyCatcher system, originally developed by the 2013 team of <a target="_blank"href="http://parts.igem.org/Part:BBa_K1159200">TU-Munich</a> was improved and adapted to secretion in <i>Bacillus subtilis</i>. (II) Secretion and interaction of SpyTag-SpyCatcher system was <a href="#results" class="hashlink">demonstrated</a>. (III) 8 <a href="#basic" class="hashlink">basic BioBrick parts</a> were improved and 8 <a href="#composite" class="hashlink">composite parts</a> were generated and evaluated.</p> |
</figure> | </figure> | ||
</div> | </div> | ||
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<div class="contentbox"> | <div class="contentbox"> | ||
<h1 id="secretion" class="box-heading">Short Description</h1> | <h1 id="secretion" class="box-heading">Short Description</h1> | ||
− | In combining <i>Bacillus subtilis</i> powerful secretion capacity with Peptidosomes as a new platform for functional co-cultivation we aim to produce multiprotein complexes. Various strains - each secreting distinct proteins of interest - can be cultivated in one reaction hub while being physically separated. In this part of EncaBcillus we study extracellular protein interaction mediated by the SpyTag/SpyCatcher system. This set-up bears the potential for an effective production of customizable biomaterials or enzyme complexes.</p> | + | <p>In combining <i>Bacillus subtilis</i> powerful secretion capacity with Peptidosomes as a new platform for functional co-cultivation we aim to produce multiprotein complexes. Various strains - each secreting distinct proteins of interest - can be cultivated in one reaction hub while being physically separated. In this part of EncaBcillus we study extracellular protein interaction mediated by the SpyTag/SpyCatcher system. This set-up bears the potential for an effective production of customizable biomaterials or enzyme complexes.</p> |
</div> | </div> | ||
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<div class="contentbox"> | <div class="contentbox"> | ||
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<h1 id="background" class="box-heading">Background</h1> | <h1 id="background" class="box-heading">Background</h1> | ||
− | <p>Efficient and low cost production of valuable natural compounds, like proteins, has developed into a leading industry. Starting from the choice of a suitable production host to the establishment of a profitable downstream process every step is constantly optimized to increase overall yields. </p> | + | <p>Efficient and low cost production of valuable natural compounds, like proteins, has developed into a leading industry. Starting from the choice of a suitable production host to the establishment of a profitable downstream process, every step is constantly optimized to increase overall yields. </p> |
<p>When it comes to choose the production host, <i>Bacillus subtilis</i> is particularly interesting: the Gram-positive model organism can be easily genetically modified and has powerful secretion capacities.<a target="_blank"href="https://www.researchgate.net/profile/Reindert_Nijland/publication/23656731_Optimization_of_Protein_Secretion_by_Bacillus_subtilis/links/09e415136142e1554c000000/Optimization-of-Protein-Secretion-by-Bacillus-subtilis.pdf">[1]</a></p> | <p>When it comes to choose the production host, <i>Bacillus subtilis</i> is particularly interesting: the Gram-positive model organism can be easily genetically modified and has powerful secretion capacities.<a target="_blank"href="https://www.researchgate.net/profile/Reindert_Nijland/publication/23656731_Optimization_of_Protein_Secretion_by_Bacillus_subtilis/links/09e415136142e1554c000000/Optimization-of-Protein-Secretion-by-Bacillus-subtilis.pdf">[1]</a></p> | ||
− | <p>In this part of EncaBcillus we aimed at making use of <i> B. subtilis</i> native advantages and | + | <p>In this part of EncaBcillus we aimed at making use of <i> B. subtilis</i> native advantages and combining them with Peptidosomes – a new innovative platform for functional co-cultivation. There is a possibility to create multiple Peptidosomes, each encapsulating one specific strain that secrets a protein of interest. By doing so, the production of multiprotein complexes could be easily achieved in one reaction hub with different subpopulations. This system is illustrated in Figure 1. </p> |
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<img src="https://static.igem.org/mediawiki/2017/e/e5/T--TU_Dresden--secretion--Background.png" | <img src="https://static.igem.org/mediawiki/2017/e/e5/T--TU_Dresden--secretion--Background.png" | ||
alt="Charting " class="zoom"> | alt="Charting " class="zoom"> | ||
− | <figcaption><b>Figure 1: Production of multiprotein complexes with Peptidosomes.</b> Depicted are two different strains of <i>B. subtilis</i> each producing a distinct protein of interest, that is either fused with SpyTag or mini. SpyCatcher. After diffusion | + | <figcaption><b>Figure 1: Production of multiprotein complexes with Peptidosomes.</b> Depicted are two different strains of <i>B. subtilis</i> each producing a distinct protein of interest, that is either fused with SpyTag or mini. SpyCatcher. After diffusion into the medium outside of the Peptidosomes, a covalent bonding of the proteins is mediated. </figcaption> |
</figure> | </figure> | ||
<p>To ensure the assembly of proteins outside of the Peptidosomes we further characterized the SpyTag/SpyCatcher system. Theses functional units can be attached to any protein of interest and upon secretion will result in a covalent isopeptide bond between the SpyTag/SpyCatcher partners.<a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/28230977">[2]</a> | <p>To ensure the assembly of proteins outside of the Peptidosomes we further characterized the SpyTag/SpyCatcher system. Theses functional units can be attached to any protein of interest and upon secretion will result in a covalent isopeptide bond between the SpyTag/SpyCatcher partners.<a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/28230977">[2]</a> | ||
− | The system originates from <i>Streptococcus pyogenes</i> and | + | The system originates from <i>Streptococcus pyogenes</i> and still remains under constant developments.<a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/22366317>[3]</a> In our project we applied codon-adapted <i> B. subtilis</i> specific tags and a SpyCatcher that is reduced in length to enhance it´s usability when translationally fused to a protein of interest. Thus, decreasing the chances of the tag interfering with overall protein folding.<a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/24161952">[4]</a></p> |
<p>To demonstrate the applicability of both tags we fused them to a green (sfGFP) and a red (mCherry) fluorescent protein, enabling an easy detectable output. (For more details please check our Design section) | <p>To demonstrate the applicability of both tags we fused them to a green (sfGFP) and a red (mCherry) fluorescent protein, enabling an easy detectable output. (For more details please check our Design section) | ||
Since a core part of this project involves secretion, we included a signal peptide in front of all our constructs. (click <a href =" https://2017.igem.org/Team:TU_Dresden/Measurement">here</a> to learn more about our Signal Peptide Toolbox).</p> | Since a core part of this project involves secretion, we included a signal peptide in front of all our constructs. (click <a href =" https://2017.igem.org/Team:TU_Dresden/Measurement">here</a> to learn more about our Signal Peptide Toolbox).</p> | ||
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<p>All of the composite parts necessary for the genetic constructs were equipped with the RFC 25 standard, cloned into pSB1C3 backbone and submitted to the registry. All cloning was done according to standard protocols and the plasmids were stored in <i>Escherichia coli</i> DH10β. All constructs were verified by sequencing.</p> | <p>All of the composite parts necessary for the genetic constructs were equipped with the RFC 25 standard, cloned into pSB1C3 backbone and submitted to the registry. All cloning was done according to standard protocols and the plasmids were stored in <i>Escherichia coli</i> DH10β. All constructs were verified by sequencing.</p> | ||
− | <p>The gene encoding the mini. SpyCatcher (<a target="_blank" href =" http://parts.igem.org/Part:BBa_K2273015">BBa_K2273015</a>) was chemically synthesized. | + | <p id="basic">The gene encoding the mini. SpyCatcher (<a target="_blank" href =" http://parts.igem.org/Part:BBa_K2273015">BBa_K2273015</a>) was chemically synthesized. |
The codon optimized SpyTag (<a target="_blank" href =" http://parts.igem.org/Part:BBa_K2273014">BBa_K2273014</a>) was generated via overlapping primers iG17P049 and G17P050 and amplified using the primers TM4487 and iG17P039. We used a <i>sfGFP</i> (<a target="_blank" href =" http://parts.igem.org/Part:BBa_K2273033">BBa_K2273033</a>) that was codon optimized for <i>Streptococcus pneumoniae</i>, which has been demonstrated to work best in <i>Bacillus subtilis</i>.<a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/23956387">[5]</a> The used <i>mCherry</i> (<a target="_blank" href =" http://parts.igem.org/Part:BBa_K2273034">BBa_K2273034</a>) was codon adapted for <i>B. subtilis</i> (Popp et al., 2017, accepted). The His-tag, necessary for protein purification was included in the reverse primers (Table 1).</p> | The codon optimized SpyTag (<a target="_blank" href =" http://parts.igem.org/Part:BBa_K2273014">BBa_K2273014</a>) was generated via overlapping primers iG17P049 and G17P050 and amplified using the primers TM4487 and iG17P039. We used a <i>sfGFP</i> (<a target="_blank" href =" http://parts.igem.org/Part:BBa_K2273033">BBa_K2273033</a>) that was codon optimized for <i>Streptococcus pneumoniae</i>, which has been demonstrated to work best in <i>Bacillus subtilis</i>.<a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/23956387">[5]</a> The used <i>mCherry</i> (<a target="_blank" href =" http://parts.igem.org/Part:BBa_K2273034">BBa_K2273034</a>) was codon adapted for <i>B. subtilis</i> (Popp et al., 2017, accepted). The His-tag, necessary for protein purification was included in the reverse primers (Table 1).</p> | ||
− | <figure class="jonathanstables" style="width:100%; | + | <figure class="jonathanstables" style="width:100%;" > |
<figcaption><b>Table 1: Overview of the constructed basic parts.</b></figcaption> | <figcaption><b>Table 1: Overview of the constructed basic parts.</b></figcaption> | ||
<table style="width:100%; display: table;"> | <table style="width:100%; display: table;"> | ||
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<div class="contentbox"> | <div class="contentbox"> | ||
− | <h1 class="box-heading">Results</h1> | + | <h1 id="results" class="box-heading">Results</h1> |
<h3>Fluorescence assay of supernatants derived from strains with single copy genes</h3> | <h3>Fluorescence assay of supernatants derived from strains with single copy genes</h3> | ||
<p>The first obstacle that we had to overcome was establishing a suitable protocol to boost the secretion capacities of our generated <i>Bacillus subitilis</i> producer strains. After initially testing various media and incubation periods we performed all experiments using 2xYT medium and an incubation time of 16 h.</p> | <p>The first obstacle that we had to overcome was establishing a suitable protocol to boost the secretion capacities of our generated <i>Bacillus subitilis</i> producer strains. After initially testing various media and incubation periods we performed all experiments using 2xYT medium and an incubation time of 16 h.</p> | ||
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alt="Figure 4: Endpoint measurement of the fluorescence from supernatants carrying our constructs and the wild type. " class="zoom"> | alt="Figure 4: Endpoint measurement of the fluorescence from supernatants carrying our constructs and the wild type. " class="zoom"> | ||
<figcaption><b>Figure 4: Endpoint measurement of the fluorescence from supernatants carrying our constructs and the wild type. </b> | <figcaption><b>Figure 4: Endpoint measurement of the fluorescence from supernatants carrying our constructs and the wild type. </b> | ||
− | Expression of the single copy mCherry or sfGFP fusion SpyTag/SpyCather constructs (purple) was induced with 1% xylose and the supernatants were harvested after 16 h of incubation. Wild type supernatant is shown as a control (pink). Excitation wavelength for sfGFP was set to 480 nm and emission was recorded at 510 nm and for mCherry excitation wavelength was set to 585 nm and emission was recorded at 615 nm . The fluorescence was normalized by the optical density ( | + | Expression of the single copy mCherry or sfGFP fusion SpyTag/SpyCather constructs (purple) was induced with 1% xylose and the supernatants were harvested after 16 h of incubation. Wild type supernatant is shown as a control (pink). Excitation wavelength for sfGFP was set to 480 nm and emission was recorded at 510 nm and for mCherry excitation wavelength was set to 585 nm and emission was recorded at 615 nm. The fluorescence was normalized by the optical density (OD<sub>600</sub>). Graph shows mean values and standard deviations of at least two biological and three technical replicates. |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
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<p></p> | <p></p> | ||
<p></p> | <p></p> | ||
− | <p>To check the efficiency of the secretion process, we also determined the | + | <p>To check the efficiency of the secretion process, we also determined the fluorescence of the cells. The supernatant was separated from the cells by centrifugation.</p> |
<p>The pelleted cells were resuspended in fresh medium, immediately followed by a fluorescence measurement (assuming the recorded fluorescence exclusively derives from FPs still inside the cells). We compared the relative fluorescence of both samples and also included untreated 16h old cell culture in this assay.</p> | <p>The pelleted cells were resuspended in fresh medium, immediately followed by a fluorescence measurement (assuming the recorded fluorescence exclusively derives from FPs still inside the cells). We compared the relative fluorescence of both samples and also included untreated 16h old cell culture in this assay.</p> | ||
<p>Figure 5 shows data generated for one strain (mCherry-mini. SpyCatcher) as an example. While the relative fluorescence in the unprocessed cell culture and the supernatant do not differ significantly, the emission of the fresh-resuspended cells show a clear drop of fluorescence (comparable to the wild-type level). </p> | <p>Figure 5 shows data generated for one strain (mCherry-mini. SpyCatcher) as an example. While the relative fluorescence in the unprocessed cell culture and the supernatant do not differ significantly, the emission of the fresh-resuspended cells show a clear drop of fluorescence (comparable to the wild-type level). </p> | ||
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alt="Figure 6: Endpoint measurement of the fluorescence from supernatants. " class="zoom"> | alt="Figure 6: Endpoint measurement of the fluorescence from supernatants. " class="zoom"> | ||
<figcaption><b>Figure 6: Endpoint measurement of the fluorescence from supernatants. </b> | <figcaption><b>Figure 6: Endpoint measurement of the fluorescence from supernatants. </b> | ||
− | Expression of the multi copy mCherry or sfGFP fusion constructs (purple) was induced with 1% Xylose and the supernatants were harvested after 16 h of incubation. Wild type supernatant is shown as a control (pink). Excitation wavelength for sfGFP was set to 480 nm and emission was recorded at 510 nm and for mCherry excitation wavelength was set to 585 nm and emission was recorded at 615 nm . The fluorescence was normalized over the optical density ( | + | Expression of the multi copy mCherry or sfGFP fusion constructs (purple) was induced with 1% Xylose and the supernatants were harvested after 16 h of incubation. Wild type supernatant is shown as a control (pink). Excitation wavelength for sfGFP was set to 480 nm and emission was recorded at 510 nm and for mCherry excitation wavelength was set to 585 nm and emission was recorded at 615 nm . The fluorescence was normalized over the optical density (OD<sub>600</sub>). Graphs show mean values and standard deviations of at least two biological and three technical replicates. |
</figcaption> | </figcaption> | ||
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alt="Figure 7: Supernatants of <i>B. subtilis</i> cultures excited with blue light." | alt="Figure 7: Supernatants of <i>B. subtilis</i> cultures excited with blue light." | ||
class="makeresponsive zoom"> | class="makeresponsive zoom"> | ||
− | <figcaption><b>Figure 7: Supernatants of <i>B. subtilis</i> cultures excited with blue light.</b> | + | <figcaption><b>Figure 7: Supernatants of <i><b>B. subtilis</b></i> cultures excited with blue light.</b> |
Wild-type supernatant (left) and a SpyTag-sfGFP secreting strain (middle and right). The expression of the multi-copy sfGFP was induced with 1% Xylose and the supernatant was harvested after 16 h of incubation. The fluorescence was induced with a “Dark Reader Transilluminator”. | Wild-type supernatant (left) and a SpyTag-sfGFP secreting strain (middle and right). The expression of the multi-copy sfGFP was induced with 1% Xylose and the supernatant was harvested after 16 h of incubation. The fluorescence was induced with a “Dark Reader Transilluminator”. | ||
</figcaption> | </figcaption> | ||
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alt="Figure 8: Supernatants of <i>B. subtilis</i> cultures." | alt="Figure 8: Supernatants of <i>B. subtilis</i> cultures." | ||
class="makeresponsive zoom"> | class="makeresponsive zoom"> | ||
− | <figcaption><b>Figure 8: Supernatants of <i>B. subtilis </i>cultures.</b> | + | <figcaption><b>Figure 8: Supernatants of <i><b>B. subtilis</b></i> cultures.</b> |
Wild-type supernatant (left) and a mCherry-mini. SpyCatcher secreting strain (right). The expression of the multi-copy mCherry was induced with 1% Xylose and the supernatant was harvested after 16 h of incubation. | Wild-type supernatant (left) and a mCherry-mini. SpyCatcher secreting strain (right). The expression of the multi-copy mCherry was induced with 1% Xylose and the supernatant was harvested after 16 h of incubation. | ||
</figcaption> | </figcaption> | ||
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<h3>Emission spectra:</h3> | <h3>Emission spectra:</h3> | ||
<p>The emission spectra of the supernatants containing fusion protein producing cultures were analysed to prove that the measured fluorescence’s originates from sfGFP or mCherry. In Figure 9 the emission spectra of the sfGFP-fusion protein is clearly visible, having an emission peak at ca. 510 nm. The mCherry-fusion protein displays the correct emission spectrum as well, having a peak at 615 nm. </p> | <p>The emission spectra of the supernatants containing fusion protein producing cultures were analysed to prove that the measured fluorescence’s originates from sfGFP or mCherry. In Figure 9 the emission spectra of the sfGFP-fusion protein is clearly visible, having an emission peak at ca. 510 nm. The mCherry-fusion protein displays the correct emission spectrum as well, having a peak at 615 nm. </p> | ||
− | <p>The 2xYT medium that was used for cultivation shows | + | <p>The 2xYT medium that was used for cultivation shows an overall high auto-fluorescence when sfGFP wavelengths were measured. As already mentioned before, this influences the total fluorescent emissions of the supernatant. Likewise, it needs to be considered that the wild-type supernatant has a lower fluorescence than the medium, suggesting a quenching effect caused by <i>B. subtilis</i>. On the other hand, only very low auto-fluorescents or quenching effects could be observed when the mCherry spectrum was recorded. Thus, we decided to carry on with mCherry constructs to prove the applicability of the SpyTag/SpyCatcher systems. </p> |
<figure class="makeresponsive" > | <figure class="makeresponsive" > | ||
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<p>By running SDS-PAGE with the purified supernatants of mCherry fusion protein producing cultures, we could prove the functionality of the SpyTag/SpyCatcher partners. In Figure 10 it is clearly visible, that the supernatants from the production strains (lane 4 and 6) contain protein bands of high concentration that the wild-type (lane 2) lacks. These proteins band are showing up again in the elution after purification, proving that the bands are referring to the his-tagged target proteins, mCherry-mini. SpyCatcher having a weight of 36,6 kDa and mCherry-SpyTag weighting 31,9 kDa.</p> | <p>By running SDS-PAGE with the purified supernatants of mCherry fusion protein producing cultures, we could prove the functionality of the SpyTag/SpyCatcher partners. In Figure 10 it is clearly visible, that the supernatants from the production strains (lane 4 and 6) contain protein bands of high concentration that the wild-type (lane 2) lacks. These proteins band are showing up again in the elution after purification, proving that the bands are referring to the his-tagged target proteins, mCherry-mini. SpyCatcher having a weight of 36,6 kDa and mCherry-SpyTag weighting 31,9 kDa.</p> | ||
− | <p> Surprisingly the supernatant with mCherry-SpyTag contained a band of the wrong weight, and the mCherry-mini. SpyCatcher even three bands the wrong weight, indicating accumulation or degradation of the protein of interest.</p> | + | <p> Surprisingly, the supernatant with mCherry-SpyTag contained a band of the wrong weight, and the mCherry-mini. SpyCatcher even three bands the wrong weight, indicating accumulation or degradation of the protein of interest.</p> |
− | <p> | + | <p>Nonetheless, upon combining two supernatants and incubating for 4 h, a new protein band with ca. 70 kDA shows up (lane 8 and 9). This is the definite proof, that the mini. SpyCatcher and the Spytag are functional and mediate the covalent bonding of the mCherry constructs. </p> |
<figure class="makeresponsive"; style="padding-left:15%; padding-right:15%" > | <figure class="makeresponsive"; style="padding-left:15%; padding-right:15%" > | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td><a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/ | + | <td><a target="_blank" href =" https://www.ncbi.nlm.nih.gov/pubmed/22366317">[3]</a></td> |
<td>Zakeri et. All (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. <i>Applied Microbiology and Biotechnology</i>.</td> | <td>Zakeri et. All (2012) Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. <i>Applied Microbiology and Biotechnology</i>.</td> | ||
</tr> | </tr> |
Latest revision as of 15:50, 13 December 2017