Part:BBa_K3002000
Spectinomycin Resistance for Chlamydomonas reinhardtii (Phytobrick)
This basic part contains the coding sequence of a spectinomycin resistance (B3-B5) and was built as a part of the Kaiser Collection. Combined with a promoter (e.g. BBa_K3002003 (PAR promoter (A1-A3)), BBa_K3002034 (Tub2 promoter (A1-A3)), or BBa_K3002036 (PSAD promoter (A1-A3))) and a terminator (e.g. BBa_K3002006 (RPL23 terminator)), this level 0 construct mediates resistance to spectinomycin. It is used as a selection marker in Chlamydomonas and is, therefore, a mandatory component of every level 2 construct.
Both MUT-PETase and MHETase show cytosolic expression and secretion when containing the cCA secretion signal. For successful expression and transformation, a reliable selection marker is needed. The aadA coding sequence allows the selection of positive transformants on spectinomycin.
MUT-PETase destined for secretion gets stuck inside the cell. (a) 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. (b) Seven days old cultures of transformants generated with the construct shown in (a) 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. (c) Whole-cell proteins of UVM4 cells transformed with construct L2C (BBa_K3002202) shown in (a) were analyzed by immuno-blotting using an anti-HA antibody. Transformant A27 generated with construct L2A (BBa_K3002200) (Figure 4a) and UVM4 were used as positive and negative controls, respectively. The white arrow indicates MUT-PETase. (d) Immunfluorescence analysis of transformants 17 and 27 using an anti-HA antibody. DAPI staining was also performed. UVM4 cells served as control.
Analysis of secreted enzymes of transformant N6 transformed with construct AI. (b) Clones generated with transformant N6 (BBa_K3002213) (Figure 8) and construct L2AI (a) (BBa_K3002234) were grown in TAP medium for four 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 C12 (BBa_K3002202) introduced in Figure 5, served as positive controls. The black arrow points to MHETase, the white arrow to MUT-PETase.
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
Usage and Biology
Team UVU-Utah-2, 2024
We obtained this part in the Chlamydomonas MoClo kit as described in Crozet et al. (2018). We utilized and characterized this part in composite part K5078004. Please check out the documentation for that composite part for more details!
Both MUT-PETase and MHETase show cytosolic expression and secretion when containing the cCA secretion signal. For successful expression and transformation, a reliable selection marker is needed. The aadA coding sequence allows the selection of positive transformants on spectinomycin.
MUT-PETase destined for secretion gets stuck inside the cell. (a) 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. (b) Seven days old cultures of transformants generated with the construct shown in (a) 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. (c) Whole-cell proteins of UVM4 cells transformed with construct L2C (BBa_K3002202) shown in (a) were analyzed by immuno-blotting using an anti-HA antibody. Transformant A27 generated with construct L2A (BBa_K3002200) (Figure 4a) and UVM4 were used as positive and negative controls, respectively. The white arrow indicates MUT-PETase. (d) Immunfluorescence analysis of transformants 17 and 27 using an anti-HA antibody. DAPI staining was also performed. UVM4 cells served as control.
Analysis of secreted enzymes of transformant N6 transformed with construct AI. (b) Clones generated with transformant N6 (Figure 8) and construct L2AI (a) (BBa_K3002234) were grown in TAP medium for four 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 C12 introduced in Figure 5, served as positive controls. The black arrow points to MHETase, the white arrow to MUT-PETase.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 586
Illegal NgoMIV site found at 769
Illegal NgoMIV site found at 879 - 1000COMPATIBLE WITH RFC[1000]
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