Part:BBa_K3002127
Level 1 – Hygromycin Resistance Gene for Chlamydomonas reinhardtii (Phytobrick)
This composite part contains the PSAD-promoter (BBa_K3002001) in combination with the PSAD-Terminator (BBa_K3002002) and the coding sequence of the Hygromycin resistance (BBa_K3002013) plus the MoClo connectors for positions B2-B3 (BBa_K3002303) and B5-B6 (BBa_K3002305). This part mediates resistance to hygromycin.
For successful expression and transformation, a reliable selection marker is needed. The hygromycin cassette allows the selection of positive transformants on hygromycin and can be used for a Co-transformation together with aadA construct. The MUT-PETase and MHETase are both seen after the co-transformation. An ARS secretion signal is upstream to the MUT-PETase gene and the cCA secretion signal upstream to the MHETase gene. The MUT-PETase is crucial for the degradation of PET into terephthalic acid and ethylene glycol.
<img src="https://2019.igem.org/wiki/images/7/7d/T--TU_Kaiserslautern--resultsFigure12.svg"/>
Analysis of secreted enzymes of transformant N6 transformed with construct AI. (b) Clones generated with transformant N6 (Figure 8) and construct L2AI (<a href="https://parts.igem.org/Part:BBa_K3002234">BBa_K3002234</a>) (a) 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.
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
- 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 1270
Illegal NgoMIV site found at 1452 - 1000COMPATIBLE WITH RFC[1000]
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