Part:BBa_K3002214
L2 spectinomycin resistance + GLE_Mut-PETase_SP20HA + cCA_MHETase_SP20HA
This composite part contains a spectinomycin resistance (BBa_K3002102), the mutant PETase with the secretion signal GLE (BBa_K3002110) and the MHETase with the cCA (BBa_K3002114), both fused with an SP20HA-tag for easy detection via HA-antibody and enhanced secretion.
The construct (LO) 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 and PETase. (a) Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase equipped with the secretion signals (BBa_K3002212, BBa_K3002213, BBa_K3002214) 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 (BBa_K3002202) and A27 (BBa_K3002200) introduced in Figures 4 and 5, respectively, served as positive controls. The black arrow points to MHETase, the white arrow to MUT-PETase.
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)(BBa_K3002202, BBa_K3002210, BBa_K3002211), 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 (BBa_K3002202, BBa_K3002210, BBa_K3002211, BBa_K3002212, BBa_K3002213, BBa_K3002214) 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.
Verification of secretion of MHETase and MUT-PETase into the medium. Transformants generated with constructs M, N, and O (BBa_K3002212, BBa_K3002213, BBa_K3002214)(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.
Quantification of secreted MHETase and MUT-PETase. (a) Transformants generated with constructs C, J, M, N, and O (BBa_K3002202, BBa_K3002208, BBa_K3002212, BBa_K3002213, BBa_K3002214)(Figure 8) 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. Whole-cell extracts of strain B1-TIG-HA for which concentrations of the HA-tagged TIG protein are known are loaded next to the lyophilized supernatants. The black arrow points to MHETase, the white arrows to MUT-PETase. The supernatant of a culture with the UVM4 strain were loaded as negative control. (b) Maximum cell densities, doubling times, daily growth rates, yields of MHETase and PETase and daily productivity of both combined were calculated for the transformant lines indicated.
The Chlamy Yummy Project Collection
We are proud to present our MoClo part collection for C. reinhardtii - the Chlamy Yummy project collection.
These 67 parts are all parts used during our project and were specifically designed and codon optimized for Chlamydomonas. Among them are basic parts (L0) of a novel mutant of the PETase (BBa_K3002014), the wildtype PETase and MHETase as well as a variety of functional composite parts (L1+2). Containing different tags as well as selection markers, this collection serves as a perfect base for plastic degradation projects with Chlamydomonas. These parts were tested and optimized thoroughly and we can guarantee that they work 100%. 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 Chlamy Yummy 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. Visit our parts site to get an overview over all parts.
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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