Revision as of 23:00, 13 December 2019

Hygromycin Resistance for Chlamydomonas reinhardtii (Phytobrick)

This basic part contains the coding sequence of a hygromycin 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 hygromycin.

Both MUT-PETase and MHETase show secretion when containing the cCA or ARS secretion signal in combination with the SP20-tag. For successful expression and transformation, a reliable selection marker is needed. The hygromycin coding sequence allows the selection of positive transformants on hygromycin and can be used for a Co-transformation together with a second antibiotic resistance.

Overview of different level 2 MoClo constructs. We designed 35 different level 2 constructs by using the modular cloning system (MoClo) and transformed these into Chlamydomonas reinhardtii. These constructs contain promoters (PPSAD, PAR, PTub2), terminators (PSADter, RPL23ter, Tub2ter), and the coding sequences for selection markers (aadA, Hygro), tags (HA, His, SP20-HA, SP20-His), secretion signals (cCA, ARS, GLE) and the enzymes MHETase, wild-type PETase (WT-PETase), mutated PETase (Mut-PETase) and the mutated PETase from the iGEM team TJUSLS China 2016 (Mutate M).

Analysis of secreted enzymes of transformant N6 (BBa_K3002213) transformed with construct AI. (b) Clones generated with transformant N6 (Figure 8) and construct L2AI(BBa_K3002234) (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 (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

Sequence and Features