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Revision as of 23:15, 18 October 2019
![](https://static.igem.org/mediawiki/2019/8/81/T--Evry_Paris-Saclay--GoldenGate.jpg)
Abstract
- Overview
- A Type IIS RFC[10] Loop assembly system for Yarrowia lipolytica
- The Loop assembly technique
- Conclusions
- References
Overview
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.
We welcome the iGEM initiative to fully support Type IIS parts that adhere to the MoClo/ PhytoBricks and Loop Type IIS assembly standards for the first time in the 2019 Competition (https://2019.igem.org/Competition/New/Type_IIS). In this framework, we designed a Loop assembly system dedicated to our chassis, the oleaginous yeast Yarrowia lipolytica.
A Type IIS RFC[10] 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[10]-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 [9] allow random integrations in Yarrowia lipolytica Po1d strain JMY195 [10] or at a zeta docking platform in Po1d derivative strains like JMY2033 [11] which has the zeta platform at the ura3-302 locus or JMY1212 [12] which has the zeta platform at the leu2-270 locus.
- The URA3 auxotrophic selection marker [13] (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. 4.1.1.23) 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 [13], LYS5 [14] or HygR [13].
- 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.
![](https://static.igem.org/mediawiki/2019/5/57/T--Evry_Paris-Saclay--Figure1.png)
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.
![](https://static.igem.org/mediawiki/2019/e/eb/T--Evry_Paris-Saclay--Figure2.png)
Table 1. Different possible Loop Type IIS cloning sites.
Part name | Sequence with BsaI and SapI sites highlighted | Part number | |
Loop Alpha-A | GCTCTTCAATGAGGAGTGAGACC | ![]() |
BBa_K2983010 |
Loop F-Beta | GGTCTCACGCTAGCATGAAGAGC | ![]() |
BBa_K2983011 |
Loop Beta-A | GCTCTTCAGCAAGGAGTGAGACC | ![]() |
BBa_K2983012 |
Loop F-Gamma | GGTCTCACGCTATACTGAAGAGC | ![]() |
BBa_K2983013 |
Loop Gamma-A | GCTCTTCATACAGGAGTGAGACC | ![]() |
BBa_K2983014 |
Loop F-Epsilon | GGTCTCACGCTACAGTGAAGAGC | ![]() |
BBa_K2983015 |
Loop Epsilon-A | GCTCTTCACAGAGGAGTGAGACC | ![]() |
BBa_K2983016 |
Loop F-Omega | GGTCTCACGCTAGGTTGAAGAGC | ![]() |
BBa_K2983017 |
Loop A-alpha | GGTCTCAGGAGAATGTGAAGAGC | ![]() |
BBa_K2983018 |
Loop Omega-B | GCTCTTCAGGTATACTTGAGACC | ![]() |
BBa_K2983019 |
Loop B-Alpha | GGTCTCATACTAATGTGAAGAGC | ![]() |
BBa_K2983020 |
Loop Omega-C | GCTCTTCAGGTAAATGTGAGACC | ![]() |
BBa_K2983021 |
Loop C-Alpha | GGTCTCAAATGAATGTGAAGAGC | ![]() |
BBa_K2983022 |
Loop Omega-E | GCTCTTCAGGTAGCTTTGAGACC | ![]() |
BBa_K2983023 |
Loop E-Alpha | GGTCTCAGCTTAATGTGAAGAGC | ![]() |
BBa_K2983024 |
Loop Omega-F | GCTCTTCAGGTACGCTTGAGACC | ![]() |
BBa_K2983025 |
Based on the general architecture of our Yarrowia lipolytica Loop assembly platform (Figure 1), we designed the pOdd-like (Table 3) and pEven-like 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 pOdd-like.
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 pEven-like 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 |
Table 3. Yarrowia lipolytica Loop assembly plasmids pEven-like 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 |