Revision as of 13:47, 13 December 2019

Level 1 � Hygromycin resistance + Tub2 Promoter + Tub2 Terminator

This composite part contains the Tub2-promoter (BBa_K3002011) in combination with the Tub2-terminator (BBa_K3002012), 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).

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 different promotor and terminator, compared to the one for the coding sequence, prevent repetition in the DNA which can decrease the expression of the constructs. The ligation of the resistance cassette was successful as well as the secretion of the substrate.

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 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. Visit our parts site to get an overview over all parts.

Sequence and Features