Part:BBa_K3002210
L2 spectinomycin resistance + ARS_Mut-PETase + cCA_MHETase
The construct encodes the secretion signal ARS in front of the MUT-PETase gene and a cCA secretion signal upstream to the MHETase gene. As selection marker an aadA cassette is used. Constructs encoding the ARS secretion signal upstream to the MUT-PETase gene show no detectable secretion of said protein. The construct containing ARS leads in comparison to the other secretion signals to the lowest yield of MHETase. Both enzymes are crucial for the degradation of PET into its terephthalic acid and ethylene glycol.
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).
The SP20 module increases the efficiency of protein secretion. (a) Level 2 MoClo constructs harboring the aadA selection marker, and the coding sequences for MUT-PETase and MHETase equipped with the secretion signals introduced in Figure 6. The constructs contain the coding sequence for a conventional 3xHA tag (C, K, L), or the 3xHA tag preceded by a SP20 tag to enhance glycosylation (M, N, O). See Figure 1 for the description of other parts. (b) UVM4 transformants containing the constructs C, K, L and M, N, O were grown in TAP medium for seven 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 A27 introduced in Figures 4, served as positive control. The black arrow points to MHETase, the white arrow to MUT-PETase and the grey arrow to RPL1 (chloroplast ribosomal 50S protein L1). The RPL1 antibody was used to detect contamination from intracellular proteins.
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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 2401
Illegal EcoRI site found at 5303
Illegal PstI site found at 3491
Illegal PstI site found at 4464
Illegal PstI site found at 6645
Illegal PstI site found at 6969
Illegal PstI site found at 7312
Illegal PstI site found at 8122 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 2401
Illegal EcoRI site found at 5303
Illegal NheI site found at 2665
Illegal NheI site found at 5567
Illegal PstI site found at 3491
Illegal PstI site found at 4464
Illegal PstI site found at 6645
Illegal PstI site found at 6969
Illegal PstI site found at 7312
Illegal PstI site found at 8122
Illegal NotI site found at 6980 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 2401
Illegal EcoRI site found at 5303
Illegal BglII site found at 7890 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 2401
Illegal EcoRI site found at 5303
Illegal PstI site found at 3491
Illegal PstI site found at 4464
Illegal PstI site found at 6645
Illegal PstI site found at 6969
Illegal PstI site found at 7312
Illegal PstI site found at 8122 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 2401
Illegal EcoRI site found at 5303
Illegal PstI site found at 3491
Illegal PstI site found at 4464
Illegal PstI site found at 6645
Illegal PstI site found at 6969
Illegal PstI site found at 7312
Illegal PstI site found at 8122
Illegal NgoMIV site found at 1401
Illegal NgoMIV site found at 1584
Illegal NgoMIV site found at 1694
Illegal NgoMIV site found at 3226
Illegal NgoMIV site found at 3253
Illegal NgoMIV site found at 4904
Illegal NgoMIV site found at 6578 - 1000COMPATIBLE WITH RFC[1000]
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