YL-pOdd6 belongs to a collection of parts that compose the Loop assembly system dedicated to the oleaginous yeast Yarrowia lipolytica (see full description bellow). This system makes fast and efficient cloning possible by Golden Gate. It offers modularity for assembling complex genetic circuits and their subsequent transfer and integration into the Yarrowia lipolytica genome.
YL-pOdd6 presents 2 loop sites: Loop Gamma-A (BBa_K2983014) & Loop F-Omega (BBa_K2983017) and thus allows assembly of transcriptional units that will be in Position 3 at the Even Level Multi-Transcriptional units composed of 3 genes.
Usage and Biology
Golden Gate [1, 2] is a powerful molecular biology technique that allows scarless assembly of a large number of DNA fragments. It makes use of type IIS restriction enzymes, such as BsaI, BsmBI, BbsI, SapI, etc., that have the peculiarity of having a recognition site outside their cutting site. This property gives several advantages during cloning:
- It allows scarless assembly: the cutting sites can be designed so that upon digestion and ligation, the final construct has only the desired sequence without the recognition sites.
- It allows assembly of a large number of fragments in a defined order: the cutting sites can be diverse and generate several overhangs after digestion that can be ligated easily and specifically, based on complementarity.
- It allows one pot digestion and ligation: the ligation is irreversible and the final DNA molecule will persist because there is no possibility of recreating the restriction sites. Thus, during the reaction, the final construct continues to accumulate, which increases the overall cloning efficiency.
Golden Gate cloning allows great freedom in design and can employed for building custom made DNA molecules. For these reasons it was adopted by the scientific community who recognised its potential even for developing standardized and modular cloning. Thus, several Golden Gate based tool kits were constructed both for prokaryotes and eukaryotes [3-7 for example]. The recently published Loop assembly system  brings Golden Gate cloning to a higher level of creativity and modularity as it allows recursive assembly of DNA fragments.
A Type IIS RFC Loop assembly system for Yarrowia lipolytica
The general architecture of the Yarrowia lipolytica Loop assembly platform is depicted in Figure 1. It is BioBrick RFC-compatible (no illegal EcoRI, XbaI, SpeI, PstI, or NotI site) and has the following features:
- Two Zeta sequences, Zeta Up (BBa_K2983000) and Zeta Down (BBa_K2983001), are flanking the platform. Zeta sequences  allow random integrations in Yarrowia lipolytica Po1d strain JMY195  or at a zeta docking platform in Po1d derivative strains like JMY2033  which has the zeta platform at the ura3-302 locus or JMY1212  which has the zeta platform at the leu2-270 locus.
- The URA3 auxotrophic selection marker  (BBa_K2983005) which is composed of the URA3 promoter (BBa_K2983002), URA3 gene (BBa_K2983003) and the URA3 terminator (BBa_K2983004). The URA3 gene encodes the orotidine 5'-phosphate decarboxylase, an enzyme (EC. 184.108.40.206) that catalyzes the decarboxylation of orotidine monophosphate to uridine monophosphate in the pyrimidine ribonucleotide synthesis pathway. In the absence of this enzyme, the cells are able to grow only if uracil or uridine is supplemented in the media. The Yarrowia lipolytica Loop assembly platform having this auxotrophic selection marker needs to be used in Δura strains.
- Two traditional cloning sites (BamHI and HindIII) are flanking the URA3 auxotrophic selection marker to allow, if needed, changing it to other selection markers like LEU2 , LYS5  or HygR .
- The Loop Type IIS cloning sites (triangles in Figure 1, see below for detailed information) and two SfiI sites in between to allow, if needed, the insertion of E. coli cloning selection markers like LacZalpha (BBa_K2448003) or reporter RFP (BBa_J04450) expression cassettes.
The Loop Type IIS cloning sites (triangles above) are a combination of BsaI and SapI restriction sites each with different cutting sites that generate well defined overhangs (circles in Figure 1, see Figure 2 for more details). A total of 50 combinations are theoretically possible and some relevant examples are listed in Table 1.
|Table 1. Different possible Loop Type IIS cloning sites.|
|Part name||Sequence||Part number|
By an ingenious combination of the two Loop sites, Pollak et al.  developed a set of vectors that allow assembly of individual parts: Promoters, 5’UTR, CDS, and Terminators (Level 0 parts) into Transcriptional units (Level 1 or Odd Level parts) and further on into Multi-Transcriptional units (Level 2 or Even Level parts) and even Multi-Multi-Transcriptional units (Level 3 or Odd Level parts).
Based on the general architecture of our Yarrowia lipolytica Loop assembly platform (Figure 1), we designed the YL-pOdd (Table 3) and YL-pEven plasmids (Table 4) that allow the same modularity for the assembly of complex genetic circuits and further are able to integrate into the oleaginous yeast genome.
|Table 2. Yarrowia lipolytica Loop assembly plasmids YL-pOdd.|
|Part name||Loop sites||Part number|
|YL-pOdd1||Loop Alpha-A & Loop F-Beta||BBa_K2983030|
|YL-pOdd2||Loop Beta-A & Loop F-Gamma||BBa_K2983031|
|YL-pOdd3||Loop Gamma-A & Loop F-Epsilon||BBa_K2983032|
|YL-pOdd4||Loop Epsilon-A & Loop F-Omega||BBa_K2983033|
|Table 3. Yarrowia lipolytica Loop assembly plasmids YL-pEven that allow assembly of 4 genes Multi-Transcriptional units.|
|Part name||Loop sites||Part number|
|YL-pEven1||Loop A-Alpha & Loop Omega-B||BBa_K2983036|
|YL-pEven2||Loop B-Alpha & Loop Omega-C||BBa_K2983037|
|YL-pEven3||Loop C-Alpha & Loop Omega-E||BBa_K2983038|
|YL-pEven4||Loop E-Alpha & Loop Omega-F||BBa_K2983039|
In addition, we expand the initial panel of combinations of two Loop sites described by Pollak et al.  to allow assembly into Multi-Transcriptional units composed of not just 4 (as done in ) but also of 2 or 3 genes at the Even Level (Table 4).
|Table 4. Yarrowia lipolytica Loop assembly plasmids YL-pEven that allow assembly of 4 genes Multi-Transcriptional units.|
|Part name||Loop sites||Part number|
|YL-pOdd5||Loop Beta-A & Loop F-Omega||BBa_K2983034|
|YL-pOdd6||Loop Gamma-A & Loop F-Omega||BBa_K2983035|
The Loop assembly technique
The empty YL-pOdd backbones (Table 2) allow the insertion of one combination of a Promoter, a 5’UTR, a CDS and a Terminator in order to form a Transcriptional unit (Level 1 / Odd level). The assembly is made by Golden Gate using BsaI as restriction enzyme, the acceptor YL-pOdd plasmid as backbone, and the 4 different individual parts flanked by BsaI sites with compatible cutting sites from the Level 0 plasmid set as inserts (Figure 3). However, in eukaryotes the Promoter and the 5’UTR are often not clearly differentiated (since the boundary between the Promoter and the 5’UTR is not precise). Therefore, in this case, the Level 1 assembly is performed with only 3 fragments. The choice of YL-pOdd backbone to be used is dictated by the position of the gene in the multi-transcriptional unit at Level 2 (Even level):
- pOdd1: for the assembly of Transcriptional units that will be in Position 1 at the Even Level
- pOdd2: for the assembly of Transcriptional units that will be in Position 2 at the Even Level Multi-Transcriptional units composed of 3 or 4 genes
- pOdd3: for the assembly of Transcriptional units that will be in Position 3 at the Even Level Multi-Transcriptional units composed of 4 genes
- pOdd4: for the assembly of Transcriptional units that will be in Position 4 at the Even Level Multi-Transcriptional units composed of 4 genes
- pOdd5: for the assembly of Transcriptional units that will be in Position 2 at the Even Level Multi-Transcriptional units composed of 2 genes
- pOdd6: for the assembly of Transcriptional units that will be in Position 3 at the Even Level Multi-Transcriptional units composed of 3 genes
The Level 1 Transcriptional units can be assembled into Multi-Transcriptional units (Level 2 or Even Level parts) by Golden Gate using SapI as restriction enzyme (Figure 4). The choice of YL-pOdd backbone to be used is dictated by the number of Level 1 Transcriptional units to be assembled and the position in the Multi-multi-transcriptional unit at Level 3 (Odd level).
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Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BamHI site found at 304
Illegal XhoI site found at 1281
Illegal XhoI site found at 1314
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 1126
Illegal NgoMIV site found at 1554
Illegal AgeI site found at 1023
- 1000Illegal BsaI site found at 1571
Illegal BsaI.rc site found at 1537
Illegal SapI.rc site found at 1587
Illegal SapI site found at 1520