Part:BBa_K3002214
L2 spectinomycin resistance + GLE_Mut-PETase_SP20HA + cCA_MHETase_SP20HA
The construct encodes the secretion signal GLE in front of the MUT-PETase gene and a cCA secretion signal upstream to the MHETase gene. Both, the MUT-PETase gene and the MHETase gene have a SP20-tag linked behind them. As selection marker an aadA cassette is used. Since a high yield of MHETase is visible, the cCA secretion signal must be highly functional. The MUT-PETase on the other hand is barely secreted when linked with a GLE secretion signal. This is especially noticeable, when compared to a MUT-PETase that has a cCA or ARS secretion signal upstream, as both show a way higher secretion of the MUT-PETase. The secretion of the MHETase is still higher than the secretion of the MUT-PETase, irrespective of the linked secretion signal. Both enzymes are crucial for the degradation of PET into its terephthalic acid and ethylene glycol.
Effect of the SP20 module on the secretion efficiency of MHETase. (a) Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase equipped with the secretion signals introduced in Figure 6 and a C-terminal SP20 tag for enhancing glycosylation. See Figure 1 for the description of other parts. (b) UVM4 transformants containing the constructs shown in (a) 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 and A27 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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The SP20 module increases the efficiency of protein secretion. (a) Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase equipped with the secretion signals introduced in Figure 6. The constructs contain the coding sequence for a conventional 3xHA tag (C, K, L), or the 3xHA tag preceded by a SP20 tag to enhance glycosylation (M, N, O). See Figure 1 for the description of other parts. (b) UVM4 transformants containing the constructs C, K, L and M, N, O 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. Transformant A27 introduced in Figures 4, served as positive control. The black arrow points to MHETase, the white arrow to MUT-PETase and the grey arrow to RPL1 (chloroplast ribosomal 50S protein L1). The RPL1 antibody was used to detect contamination from intracellular proteins.
Identification of MHETase and MUT-PETase by LC-MS/MS. (a) Transformants generated with construct L2N (d) 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. Protein bands corresponding to those detected with the anti-HA antibody in a gel run in parallel and stained with Coomassie brilliant blue were excised, in-gel digested with trypsin and analyzed by LC-MS/MS. Peptides identified by LC-MS/MS for MHETase (green) and MUT-PETase (purple) are indicated. (b, c) Sequences of MHETase and MUT-PETase with the peptides detected by LC-MS/MS are highlighted in green and purple, respectively.
Verification of secretion of MHETase and MUT-PETase into the medium. Transformants generated with constructs M, N, and O (Figure 8) were grown in TAP medium for seven days. Cells were centrifuged and the supernatant (s) lyophilized and resuspended in 2xSDS buffer. Cell pellets (p) were also resuspended in SDS-buffer. Both fractions were analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody. The black arrow points to MHETase, the white arrow to MUT-PETase.
Growth and secretion of MUT-PETase and MHETase in CC-4533 transformant M8 in two photobioreactors. (a, b) Time course analysis of secreted MUT-PETase and MHETase in bioreactors A (a) and B (b). The cell density in bioreactor A was held at a higher cell density than that in bioreactor B. Samples were taken once or twice a day starting at 48.2 h after inoculation. Lyophilized cell-free media was resuspended in 2xSDS loading buffer and analysed by immuno-blotting using an HA-antibody. (c) Cell growth in the Bioreactors A and B at 25°C.
Purification of HA-tagged MUT-PETase and MHETase from Chlamydomonas and activity measurement against BHET by reversed-phase HPLC. (a) Affinity purification of MUT-PETase and MHETase from Chlamydomonas by anti-HA magnetic beads. Transformants M5, C12 and untransformed UVM4 were inoculated in TAP for seven days. Cultures were centrifuged and supernatants incubated with anti-HA magnetic beads for 1 h. Enzymes were purified via biomagnetic separation. Samples of the unconcentrated supernatant (S), of the washing step (W), of the eluted proteins (E) and after 96 h incubation with BHET (A.I.) were analyzed by immunoblotting using an anti-HA antibody. (b) Proteins eluted from Chlamydomonas transformant M5 (producing MUT-PETase and MHETase) and the UVM4 strain (not producing MUT-PETase and MHETase) were incubated with 1 mM BHET in sodium phosphate (NaPi) buffer at 30°C for 96 h. The standard containing 1 mM TPA, MHET and BHET dissolved in DMSO is shown on top.
Measurement of activity of MHETase and MUT-PETase from Chlamydomonas against PET by reversed-phase HPLC. 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. (a) A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. (b) The 20-fold concentrated medium of transformant M8 was incubated with PET film at 25°C for 96 h and measured with HPLC. (c) 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. (d) 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.
Activity measurement of MHETase and MUT-PETase from Chlamydomonas against BHET by reversed-phase HPLC. 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. (a) A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. (b) The 20-fold concentrated medium of M5 was incubated with BHET at 30°C for 48 h and measured by HPLC. (c) The 20-fold concentrated medium of parent strain UVM4 was incubated with BHET and measured by HPLC. (d) The glycine buffer was measured with HPLC. (e) Peak areas of the shown measurements in mAu*s.
Activity measurement of MHETase from Chlamydomonas against MHET by reversed-phase HPLC. 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. (a) A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. (b) Measurement of M5 supernatant with HPLC. (c) Measurement of UVM4 supernatant with HPLC. (d) The glycine buffer was measured with HPLC. (e) Peak areas of the shown measurements in mAu*s.
Measurement of activity of MUT-PETase and MHETase from Chlamydomonas at 25°C and 33°C by reversed-phase HPLC. 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. (a) 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 (b) and 33°C (c). Measurement of M8 medium incubated with PET at 25°C (d, e) and 33°C (f, g). measured by HPLC. The measurements shown in (d) and (e), and in (f) and (g) are the same, but the axis scaling was set to 2000 mAU in (d) and (f) and to 50 mAU in (e) and (g). Note that the shifts in retention times are due to the clogging of the HPLC.
Measurement of activity of MUT-PETase and MHETase from Chlamydomonas at 30°C and 40°C by reversed-phase HPLC. 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. (a) A standard of 1 mM TPA, MHET, and BHET dissolved in DMSO was measured by HPLC. (b, e) Measurement of M5 medium incubated with BHET at 30°C (b) and 40°C (e). (c, f) Measurement of UVM4 medium incubated with BHET at 30°C (c) and 40°C (f). (d, g) Measurement of glycine buffer incubated with BHET at 30°C (d) and 40°C (g). (h) Peak areas of the shown measurements in mAu*s.
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 EcoRI site found at 2401
Illegal EcoRI site found at 5435
Illegal PstI site found at 3503
Illegal PstI site found at 4476
Illegal PstI site found at 6777
Illegal PstI site found at 7101
Illegal PstI site found at 7444
Illegal PstI site found at 8254 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2401
Illegal EcoRI site found at 5435
Illegal NheI site found at 2665
Illegal NheI site found at 5699
Illegal PstI site found at 3503
Illegal PstI site found at 4476
Illegal PstI site found at 6777
Illegal PstI site found at 7101
Illegal PstI site found at 7444
Illegal PstI site found at 8254
Illegal NotI site found at 7112 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2401
Illegal EcoRI site found at 5435
Illegal BglII site found at 8022 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2401
Illegal EcoRI site found at 5435
Illegal PstI site found at 3503
Illegal PstI site found at 4476
Illegal PstI site found at 6777
Illegal PstI site found at 7101
Illegal PstI site found at 7444
Illegal PstI site found at 8254 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2401
Illegal EcoRI site found at 5435
Illegal PstI site found at 3503
Illegal PstI site found at 4476
Illegal PstI site found at 6777
Illegal PstI site found at 7101
Illegal PstI site found at 7444
Illegal PstI site found at 8254
Illegal NgoMIV site found at 1401
Illegal NgoMIV site found at 1584
Illegal NgoMIV site found at 1694
Illegal NgoMIV site found at 3238
Illegal NgoMIV site found at 3265
Illegal NgoMIV site found at 5036
Illegal NgoMIV site found at 6710 - 1000COMPATIBLE WITH RFC[1000]
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