Part:BBa_K3002102
Level 1 - Spectinomycin 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 Spectinomycin resistance (BBa_K3002000) plus the MoClo connectors for positions B2-B3 (BBa_K3002303) and B5-B6 (BBa_K3002305). This part mediates resistance to spectinomycin.
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 resistance cassette allows the selection of positive transformants on spectinomycin.
Expression of the enzymes MUT-PETase and MHETase in Chlamydomonas reinhardtii. (a) Level 2 MoClo construct harboring the aadA selection marker and the coding sequences for MUT-PETase, and MHETase (see Figure 1 for part description). (b) The UVM4 strain was transformed with the construct shown in (a). 11 spectinomycin-resistant transformants were inoculated in TAP and samples taken after 3 days. Extracted whole-cell proteins were analysed by SDS-PAGE and immunoblotting using an anti-HA antibody. MW – molecular weight. The black arrow represents the MHETase, the white arrow the MUT-PETase. The expression of both MHETase (~70 kDa) and MUT-PETase (~35 kDa) is visible in colonies 18, 22 and 27. The UVM4 recipient strain and a strain expressing the HA-tagged ribosomal chloroplastic 50S protein L5 (RPL5) served as negative and positive controls, respectively.
The Kaiser Collection
We are proud to present our very own MoClo part collection for C. reinhardtii - the <a href="https://2019.igem.org/Team:TU_Kaiserslautern/Part_Collection">Kaiser collection</a>.
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 <a href="https://2019.igem.org/Team:TU_Kaiserslautern/Part_Collection">part collection site</a> to get an overview over all parts of the Kaiser Collection
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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 1401
Illegal NgoMIV site found at 1584
Illegal NgoMIV site found at 1694 - 1000COMPATIBLE WITH RFC[1000]
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