Difference between revisions of "Part:BBa K3002014"
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<img src="https://2019.igem.org/wiki/images/b/b8/T--TU_Kaiserslautern--resultsFigure5.svg"/> | <img src="https://2019.igem.org/wiki/images/b/b8/T--TU_Kaiserslautern--resultsFigure5.svg"/> | ||
<p class="caption"><span class="phat">MUT-PETase destined for secretion gets stuck inside the cell. | <p class="caption"><span class="phat">MUT-PETase destined for secretion gets stuck inside the cell. | ||
− | </span><span class="accent">(a)</span> Level 2 MoClo construct harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase genes. MUT-PETase and MHETase are equipped with the secretion signal from carbonic anhydrase (cCA). See Figure 1 for the description of other parts. <span class="accent">(b)</span> Seven days old cultures of transformants generated with the construct shown in <span class="accent">(a)</span> were centrifuged and proteins in the culture medium were precipitated by TCA and analysed by immunoblotting using an anti-HA antibody. The black arrow represents MHETase. <span class="accent">(c)</span> Whole-cell proteins of UVM4 cells transformed with construct L2C shown in <span class="accent">(a)</span> were analyzed by immuno-blotting using an anti-HA antibody. Transformant A27 generated with construct L2A (Figure 4a) and UVM4 were used as positive and negative controls, respectively. The white arrow indicates MUT-PETase. <span class="accent">(d)</span> Immunfluorescence analysis of transformants 17 and 27 using an anti-HA antibody. DAPI staining was also performed. UVM4 cells served as control. | + | </span><span class="accent">(a)</span> Level 2 MoClo construct harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase genes. MUT-PETase and MHETase are equipped with the secretion signal from carbonic anhydrase (cCA). See Figure 1 for the description of other parts. <span class="accent">(b)</span> Seven days old cultures of transformants generated with the construct shown in <span class="accent">(a)</span> were centrifuged and proteins in the culture medium were precipitated by TCA and analysed by immunoblotting using an anti-HA antibody. The black arrow represents MHETase. <span class="accent">(c)</span> Whole-cell proteins of UVM4 cells transformed with construct L2C (<a href="https://parts.igem.org/Part:BBa_K3002202">BBa_K3002202</a>) shown in <span class="accent">(a)</span> were analyzed by immuno-blotting using an anti-HA antibody. Transformant A27 generated with construct L2A (<a href="https://parts.igem.org/Part:BBa_K3002200">BBa_K3002200</a>) (Figure 4a) and UVM4 were used as positive and negative controls, respectively. The white arrow indicates MUT-PETase. <span class="accent">(d)</span> Immunfluorescence analysis of transformants 17 and 27 using an anti-HA antibody. DAPI staining was also performed. UVM4 cells served as control. |
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<div class="figure"> | <div class="figure"> | ||
<img src="https://2019.igem.org/wiki/images/9/90/T--TU_Kaiserslautern--resultsFigure7.svg"/> | <img src="https://2019.igem.org/wiki/images/9/90/T--TU_Kaiserslautern--resultsFigure7.svg"/> | ||
− | <p class="caption"><span class="phat">Effect of the SP20 module on the secretion efficiency of MHETase. | + | <p class="caption"><span class="phat">Effect of the SP20 module on the secretion efficiency of MHETase and PETase. |
</span><span class="accent">(a)</span> Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase equipped with the secretion signals (<a href="https://parts.igem.org/Part:BBa_K3002212">BBa_K3002212</a>, <a href="https://parts.igem.org/Part:BBa_K3002213">BBa_K3002213</a>, <a href="https://parts.igem.org/Part:BBa_K3002214">BBa_K3002214</a>) introduced in Figure 6 and a C-terminal SP20 tag for enhancing glycosylation. See <a href="https://2019.igem.org/Team:TU_Kaiserslautern/Results">Figure 1</a> for the description of other parts. <span class="accent">(b)</span> UVM4 transformants containing the constructs shown in <span class="accent">(a)</span> were grown in TAP medium for seven days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. Transformants C12 (<a href="https://parts.igem.org/Part:BBa_K3002202">BBa_K3002202</a>) and A27 (<a href="https://parts.igem.org/Part:BBa_K3002200">BBa_K3002200</a>) introduced in Figures 4 and 5, respectively, served as positive controls. The black arrow points to MHETase, the white arrow to MUT-PETase. | </span><span class="accent">(a)</span> Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase equipped with the secretion signals (<a href="https://parts.igem.org/Part:BBa_K3002212">BBa_K3002212</a>, <a href="https://parts.igem.org/Part:BBa_K3002213">BBa_K3002213</a>, <a href="https://parts.igem.org/Part:BBa_K3002214">BBa_K3002214</a>) introduced in Figure 6 and a C-terminal SP20 tag for enhancing glycosylation. See <a href="https://2019.igem.org/Team:TU_Kaiserslautern/Results">Figure 1</a> for the description of other parts. <span class="accent">(b)</span> UVM4 transformants containing the constructs shown in <span class="accent">(a)</span> were grown in TAP medium for seven days. Cells were centrifuged and the supernatant lyophilized, resuspended in 2xSDS buffer and analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. Transformants C12 (<a href="https://parts.igem.org/Part:BBa_K3002202">BBa_K3002202</a>) and A27 (<a href="https://parts.igem.org/Part:BBa_K3002200">BBa_K3002200</a>) introduced in Figures 4 and 5, respectively, served as positive controls. The black arrow points to MHETase, the white arrow to MUT-PETase. | ||
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<img src="https://2019.igem.org/wiki/images/7/74/T--TU_Kaiserslautern--resultsFigure20.svg"/> | <img src="https://2019.igem.org/wiki/images/7/74/T--TU_Kaiserslautern--resultsFigure20.svg"/> | ||
<p class="caption"><span class="phat">Measurement of activity of MHETase and MUT-PETase from <i>Chlamydomonas</i> against PET by reversed-phase HPLC. | <p class="caption"><span class="phat">Measurement of activity of MHETase and MUT-PETase from <i>Chlamydomonas</i> against PET by reversed-phase HPLC. | ||
− | </span>Transformant M8 and parent strain CC-4533 (CliP) were inoculated in HMP medium for seven days. M8 contains construct L2M encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 13). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. <span class="accent">(a)</span> A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. <span class="accent">(b)</span> The 20-fold concentrated medium of transformant M8 was incubated with PET film at 25°C for 96 h and measured with HPLC. <span class="accent">(c)</span> The 20-fold concentrated medium of parent strain CC-4533 (CliP) was incubated with PET film at 25°C for 96 h and measured with HPLC. <span class="accent">(d)</span> The glycine buffer was measured with HPLC. The same measurements are displayed, but the scaling of the axis was set to 2000 mAU on the left and to 50 mAU on the right. | + | </span>Transformant M8 and parent strain CC-4533 (CliP) were inoculated in HMP medium for seven days. M8 contains construct L2M (<a href="https://parts.igem.org/Part:BBa_K3002212">BBa_K3002212</a>) encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 13). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. <span class="accent">(a)</span> A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. <span class="accent">(b)</span> The 20-fold concentrated medium of transformant M8 was incubated with PET film at 25°C for 96 h and measured with HPLC. <span class="accent">(c)</span> The 20-fold concentrated medium of parent strain CC-4533 (CliP) was incubated with PET film at 25°C for 96 h and measured with HPLC. <span class="accent">(d)</span> The glycine buffer was measured with HPLC. The same measurements are displayed, but the scaling of the axis was set to 2000 mAU on the left and to 50 mAU on the right. |
</p> | </p> | ||
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<img src="https://2019.igem.org/wiki/images/6/6c/T--TU_Kaiserslautern--resultsFigure21.svg"/> | <img src="https://2019.igem.org/wiki/images/6/6c/T--TU_Kaiserslautern--resultsFigure21.svg"/> | ||
<p class="caption"><span class="phat">Activity measurement of MHETase and MUT-PETase from <i>Chlamydomonas</i> against BHET by reversed-phase HPLC. | <p class="caption"><span class="phat">Activity measurement of MHETase and MUT-PETase from <i>Chlamydomonas</i> against BHET by reversed-phase HPLC. | ||
− | </span>Transformant M5 and parent strain UVM4 were inoculated in HMP medium for seven days. M5 contains construct L2M encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 8). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. <span class="accent">(a)</span> A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. <span class="accent">(b)</span> The 20-fold concentrated medium of M5 was incubated with BHET at 30°C for 48 h and measured by HPLC. <span class="accent">(c)</span> The 20-fold concentrated medium of parent strain UVM4 was incubated with BHET and measured by HPLC. <span class="accent">(d)</span> The glycine buffer was measured with HPLC. <span class="accent">(e)</span> Peak areas of the shown measurements in mAu*s. | + | </span>Transformant M5 and parent strain UVM4 were inoculated in HMP medium for seven days. M5 contains construct L2M (<a href="https://parts.igem.org/Part:BBa_K3002212">BBa_K3002212</a>) encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 8). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. <span class="accent">(a)</span> A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. <span class="accent">(b)</span> The 20-fold concentrated medium of M5 was incubated with BHET at 30°C for 48 h and measured by HPLC. <span class="accent">(c)</span> The 20-fold concentrated medium of parent strain UVM4 was incubated with BHET and measured by HPLC. <span class="accent">(d)</span> The glycine buffer was measured with HPLC. <span class="accent">(e)</span> Peak areas of the shown measurements in mAu*s. |
</p> | </p> | ||
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<img src="https://2019.igem.org/wiki/images/e/ec/T--TU_Kaiserslautern--resultsFigure22.svg"/> | <img src="https://2019.igem.org/wiki/images/e/ec/T--TU_Kaiserslautern--resultsFigure22.svg"/> | ||
<p class="caption"><span class="phat">Activity measurement of MHETase from <i>Chlamydomonas</i> against MHET by reversed-phase HPLC. | <p class="caption"><span class="phat">Activity measurement of MHETase from <i>Chlamydomonas</i> against MHET by reversed-phase HPLC. | ||
− | </span>Transformant M5 and parent strain UVM4 were inoculated in HMP medium for seven days. M5 contains construct L2M encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 8). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. Samples were incubated with 1 mM MHET at 30°C for 48 h. <span class="accent">(a)</span> A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. <span class="accent">(b)</span> Measurement of M5 supernatant with HPLC. <span class="accent">(c)</span> Measurement of UVM4 supernatant with HPLC. <span class="accent">(d)</span> The glycine buffer was measured with HPLC. <span class="accent">(e)</span> Peak areas of the shown measurements in mAu*s. | + | </span>Transformant M5 and parent strain UVM4 were inoculated in HMP medium for seven days. M5 contains construct L2M (<a href="https://parts.igem.org/Part:BBa_K3002212">BBa_K3002212</a>)encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 8). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. Samples were incubated with 1 mM MHET at 30°C for 48 h. <span class="accent">(a)</span> A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. <span class="accent">(b)</span> Measurement of M5 supernatant with HPLC. <span class="accent">(c)</span> Measurement of UVM4 supernatant with HPLC. <span class="accent">(d)</span> The glycine buffer was measured with HPLC. <span class="accent">(e)</span> Peak areas of the shown measurements in mAu*s. |
</p> | </p> | ||
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<img src="https://2019.igem.org/wiki/images/2/24/T--TU_Kaiserslautern--resultsFigure23.svg"/> | <img src="https://2019.igem.org/wiki/images/2/24/T--TU_Kaiserslautern--resultsFigure23.svg"/> | ||
<p class="caption"><span class="phat">Measurement of activity of MUT-PETase and MHETase from <i>Chlamydomonas</i> at 25°C and 33°C by reversed-phase HPLC. | <p class="caption"><span class="phat">Measurement of activity of MUT-PETase and MHETase from <i>Chlamydomonas</i> at 25°C and 33°C by reversed-phase HPLC. | ||
− | </span>Transformant M8 and parent strain CC-4533 (CliP) were inoculated in HMP medium for seven days. M8 contains construct L2M encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 13). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. Samples were incubated with PET film or MHET at 25°C or 33°C for 96 h. <span class="accent">(a)</span> A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. Measurement of M8 medium incubated with MHET at 25°C <span class="accent">(b)</span> and 33°C <span class="accent">(c)</span>. Measurement of M8 medium incubated with PET at 25°C (d, e) and 33°C (f, g). measured by HPLC. The measurements shown in <span class="accent">(d)</span> and <span class="accent">(e)</span>, and in <span class="accent">(f)</span> and <span class="accent">(g)</span> are the same, but the axis scaling was set to 2000 mAU in <span class="accent">(d)</span> and <span class="accent">(f)</span> and to 50 mAU in <span class="accent">(e)</span> and <span class="accent">(g)</span>. Note that the shifts in retention times are due to the clogging of the HPLC. | + | </span>Transformant M8 and parent strain CC-4533 (CliP) were inoculated in HMP medium for seven days. M8 contains construct L2M (<a href="https://parts.igem.org/Part:BBa_K3002212">BBa_K3002212</a>) encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 13). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. Samples were incubated with PET film or MHET at 25°C or 33°C for 96 h. <span class="accent">(a)</span> A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. Measurement of M8 medium incubated with MHET at 25°C <span class="accent">(b)</span> and 33°C <span class="accent">(c)</span>. Measurement of M8 medium incubated with PET at 25°C (d, e) and 33°C (f, g). measured by HPLC. The measurements shown in <span class="accent">(d)</span> and <span class="accent">(e)</span>, and in <span class="accent">(f)</span> and <span class="accent">(g)</span> are the same, but the axis scaling was set to 2000 mAU in <span class="accent">(d)</span> and <span class="accent">(f)</span> and to 50 mAU in <span class="accent">(e)</span> and <span class="accent">(g)</span>. Note that the shifts in retention times are due to the clogging of the HPLC. |
</p> | </p> | ||
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<img src="https://2019.igem.org/wiki/images/c/c0/T--TU_Kaiserslautern--resultsFigure24.svg"/> | <img src="https://2019.igem.org/wiki/images/c/c0/T--TU_Kaiserslautern--resultsFigure24.svg"/> | ||
<p class="caption"><span class="phat">Measurement of activity of MUT-PETase and MHETase from Chlamydomonas at 30°C and 40°C by reversed-phase HPLC. | <p class="caption"><span class="phat">Measurement of activity of MUT-PETase and MHETase from Chlamydomonas at 30°C and 40°C by reversed-phase HPLC. | ||
− | </span>Transformant M5 and parent strain UVM4 were inoculated in HMP medium for seven days. M5 contains construct L2M encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 8). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. Samples were incubated with 1 mM BHET at 30°C (left) or 40°C (right) for 96 h. <span class="accent">(a)</span> A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. <span class="accent">(b, e)</span> Measurement of M5 medium incubated with BHET at 30°C <span class="accent">(b)</span> and 40°C <span class="accent">(e)</span>. <span class="accent">(c, f)</span> Measurement of UVM4 medium incubated with BHET at 30°C <span class="accent">(c)</span> and 40°C <span class="accent">(f)</span>. <span class="accent">(d, g)</span> Measurement of glycine buffer incubated with BHET at 30°C <span class="accent">(d)</span> and 40°C <span class="accent">(g)</span>. <span class="accent">(h)</span> Peak areas of the shown measurements in mAu*s. | + | </span>Transformant M5 and parent strain UVM4 were inoculated in HMP medium for seven days. M5 contains construct L2M (<a href="https://parts.igem.org/Part:BBa_K3002212">BBa_K3002212</a>) encoding MUT-PETase and MHETase tagged with the SP20-module and secretion signal cCA (Figure 8). The cultures were centrifuged, and the supernatants concentrated 20-fold with ultracentrifuge filters and rebuffered in glycine buffer. Samples were incubated with 1 mM BHET at 30°C (left) or 40°C (right) for 96 h. <span class="accent">(a)</span> A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. <span class="accent">(b, e)</span> Measurement of M5 medium incubated with BHET at 30°C <span class="accent">(b)</span> and 40°C <span class="accent">(e)</span>. <span class="accent">(c, f)</span> Measurement of UVM4 medium incubated with BHET at 30°C <span class="accent">(c)</span> and 40°C <span class="accent">(f)</span>. <span class="accent">(d, g)</span> Measurement of glycine buffer incubated with BHET at 30°C <span class="accent">(d)</span> and 40°C <span class="accent">(g)</span>. <span class="accent">(h)</span> Peak areas of the shown measurements in mAu*s. |
</p> | </p> | ||
</div> | </div> |
Revision as of 23:10, 13 December 2019
Mutant PETase for Chlamydomonas reinhardtii (Phytobrick)
The MUT-PETase was expressed with the secretion signals cCA, ARS and GLE by C.reinhardtii. The secretion was successful in combination with the SP20 tag and worked best with the secretion signals cCA and ARS. The Mut-PETase is essential for the degradation of PET into MHET and showed activity against PET and BHET.
The Kaiser Collection
We are proud to present our very own MoClo part collection for C. reinhardtii - the Kaiser collection.
These 20 Parts are specifically designed and codon optimized for Chlamydomonas. Among them are regulatory elements, antibiotic resistances, resistance cassettes, secretion signals and tags. These parts were tested and optimized thoroughly and we can guarantee that they work 100%. With these, expression and secretion in Chlamy will be a success. Because this is a MoClo collection, the parts are highly standardized for worldwide application. The combination with other part collections works fast and easy. While in MoClo, nomenclature is a bit different from the iGEM BioBricks, it is quickly explained:
Level 0 parts are equivalent to basic parts, e.g. Promoters, coding sequences, etc.
Level 1 parts are combinations of basic parts and usually form functional transcription units.
Level 2 parts are combinations of Level 1 parts, in case you want to transfer multiple transcription units at once. For example, you can pair your gene of interest with a selection marker.
The great thing about the Kaiser Collection and MoClo is that the ligation works in a one pot, one step reaction, as the Type IIs restriction enzymes cut out their own recognition sites. This way, multiple constructs can be combined linearly in a fixed order to create complex structures. This is ensured by the standardized overlaps that assign the parts one of 10 positions in the final constructs. After trying MoClo once, you won’t go back to traditional ligation. It is incredibly easy and reliable. For this reason, we believe that our Kaiser Collection will strike a significant chord, as the future lies in standardized, easy to use methods such as MoClo. Visit our part collection site to get an overview over all parts of the Kaiser Collection
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 507
Illegal PstI site found at 1480 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 507
Illegal PstI site found at 1480 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 507
Illegal PstI site found at 1480 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 507
Illegal PstI site found at 1480
Illegal NgoMIV site found at 242
Illegal NgoMIV site found at 269 - 1000COMPATIBLE WITH RFC[1000]