Team:DTU-Denmark/Parts
Awards
Parts
This page will provide a complete overview of the 90 parts we have designed and/or used for the PHEAST project.
(Click the table Headers to sort alphabetically!)
Toolbox for Chromosomal Integration and/or Deletion in Komagataella phaffii GS115
We developed an experimentally validated state of the art CRISPR based toolbox that enables the user easy and fast genetic engineering of K. phaffii GS115. The toolbox contains a set of crRNA and their associated homology for flanking the expression cassette of interest for chromosomal integration and/or deletion in K. phaffii GS115. The present collection comprises 58 parts for integrating genes in neutral integration sites in the four chromosomes of K. phaffii GS115 or deleting genes related to methanol to formaldehyde conversion. The parts can be combined in endless ways to engineer K. phaffii GS115 in the future. The PHEAST team presents the Part Collection.
Parts from Professor Uffe Hasbro Mortensen's Lab
A CRISPR-Cas9 plasmid system designed, built and supplied by Uffe Mortensen's Lab made the foundation for this toolbox. The two plasmids, pDIV151 and pDIV153, were used. pDIV153 is a CRISPR-Cas9 plasmid with a sgRNA targeting the URA3 gene. It served as a template for constructing fragments for USER cloning containing our designed crRNAs targeting intergenic regions within the genome of K. phaffii GS115. pDIV151 is a Cas9 plasmid. It served as a receiver plasmid backbone for our designed crRNAs. For confidential matters, the sequence of the plasmids cannot be made publicly available at the iGEM registry. As a compromise, we uploaded the gRNA backbone, which will be updated to contain the entire plasmid Cas9 plasmid once published.
In total sixteen crRNAs have been designed to target neutral integration sites in the four chromosomes of K. phaffii GS115. Of those sixteen, six crRNA (BBa_K3841016-BBa_K3841021) have been experimentally validated to induce double-stranded breaks in neutral integration sites in the four respective chromosomes of K. phaffii GS115 Δku70 strain.
For knocking out the genes responsible for methanol to formaldehyde conversion, six crRNA (BBa_K3841027-BBa_K3841032) were designed to target aox1, aox2, and adh2 genes in K. phaffii GS115. All 6 were experimentally validated to cause double-stranded breaks in the respective genes.
crRNAs and Repair Templates for Deletion
| Part name | Part Type | Description |
|---|---|---|
| BBa_K3841027 | RNA | gRNA for CRISPR mediated knockout of the aox1 gene in Komagataella phaffii here refered to as crRNA_GS115_AOX1_C1 |
| BBa_K3841028 | RNA | gRNA for CRISPR mediated knockout of the aox1 gene in Komagataella phaffii here refered to as crRNA_GS115_AOX1_C2 |
| BBa_K3841029 | RNA | gRNA for CRISPR mediated knockout of the aox2 gene in Komagataella phaffii here refered to as crRNA_GS115_AOX2_C1 |
| BBa_K3841030 | RNA | gRNA for CRISPR mediated knockout of the aox2 gene in Komagataella phaffii here refered to as crRNA_GS115_AOX2_C2 |
| BBa_K3841031 | RNA | gRNA for CRISPR mediated knockout of the adh2 gene in Komagataella phaffii here refered to as crRNA_GS115_ADH2_C1 |
| BBa_K3841032 | RNA | gRNA for CRISPR mediated knockout of the adh2 gene in Komagataella phaffii here refered to as crRNA_GS115_ADH2_C2 |
| BBa_K3841033 | DNA | Repair template for AOX1 knockout |
| BBa_K3841034 | DNA | Repair template for AOX2 knockout |
| BBa_K3841035 | DNA | Repair template for ADH2 knockout |
crRNAs and Homology Arms for Integration
| Part name | Part Type | Description |
|---|---|---|
| BBa_K3841016 | RNA | gRNA for CRISPR neutral insertion site 1 in Komagataella phaffii here refered to as gRNA1_Chr_1_Cand_1 |
| BBa_K3841017 | RNA | gRNA for CRISPR neutral insertion site 2 in Komagataella phaffii here refered to as gRNA5_Chr_2_Cand_1 |
| BBa_K3841018 | RNA | gRNA for CRISPR neutral insertion site 2 in Komagataella phaffii here refered to as gRNA6_Chr_2_Cand_2 |
| BBa_K3841019 | RNA | gRNA for CRISPR neutral insertion site 3 in Komagataella phaffii here refered to as gRNA13_Chr_3_Cand_1 |
| BBa_K3841020 | RNA | gRNA for CRISPR neutral insertion site 3 in Komagataella phaffii here refered to as gRNA14_Chr_3_Cand_2 |
| BBa_K3841021 | RNA | gRNA for CRISPR neutral insertion site 4 in Komagataella phaffii here refered to as gRNA11_Chr_4_Cand_3 |
| BBa_K3841078 | RNA | gRNA for CRISPR neutral insertion site 1 in Komagataella phaffii here refered to as gRNA2_Chr_1_Cand_2 |
| BBa_K3841079 | RNA | gRNA for CRISPR neutral insertion site 1 in Komagataella phaffii here refered to as gRNA3_Chr_1_Cand_3 |
| BBa_K3841080 | RNA | gRNA for CRISPR neutral insertion site 1 in Komagataella phaffii here refered to as gRNA4_Chr_1_Cand_4 |
| BBa_K3841081 | RNA | gRNA for CRISPR neutral insertion site 2 in Komagataella phaffii here refered to as gRNA7_Chr_2_Cand_3 |
| BBa_K3841082 | RNA | gRNA for CRISPR neutral insertion site 2 in Komagataella phaffii here refered to as gRNA8_Chr_2_Cand_4 |
| BBa_K3841083 | RNA | gRNA for CRISPR neutral insertion site 3 in Komagataella phaffii here refered to as gRNA15_Chr_3_Cand_3 |
| BBa_K3841084 | RNA | gRNA for CRISPR neutral insertion site 3 in Komagataella phaffii here refered to as gRNA16_Chr_3_Cand_4 |
| BBa_K3841085 | RNA | gRNA for CRISPR neutral insertion site 4 in Komagataella phaffii here refered to as gRNA9_Chr_4_Cand_1 |
| BBa_K3841086 | RNA | gRNA for CRISPR neutral insertion site 4 in Komagataella phaffii here refered to as gRNA10_Chr_4_Cand_2 |
| BBa_K3841087 | RNA | gRNA for CRISPR neutral insertion site 4 in Komagataella phaffii here refered to as gRNA12_Chr_4_Cand_4 |
| BBa_K3841037 | DNA | Homology arm for neutral insertion site 1 in Komagataella phaffii |
| BBa_K3841038 | DNA | Homology arm for neutral insertion site 1 in Komagataella phaffii |
| BBa_K3841039 | DNA | Homology arm for neutral insertion site 1 in Komagataella phaffii |
| BBa_K3841040 | DNA | Homology arm for neutral insertion site 1 in Komagataella phaffii |
| BBa_K3841041 | DNA | Homology arm for neutral insertion site 1 in Komagataella phaffii |
| BBa_K3841042 | DNA | Homology arm for neutral insertion site 1 in Komagataella phaffii |
| BBa_K3841043 | DNA | Homology arm for neutral insertion site 1 in Komagataella phaffii |
| BBa_K3841044 | DNA | Homology arm for neutral insertion site 1 in Komagataella phaffii |
| BBa_K3841045 | DNA | Homology arm for neutral insertion site 2 in Komagataella phaffii |
| BBa_K3841046 | DNA | Homology arm for neutral insertion site 2 in Komagataella phaffii |
| BBa_K3841047 | DNA | Homology arm for neutral insertion site 2 in Komagataella phaffii |
| BBa_K3841048 | DNA | Homology arm for neutral insertion site 2 in Komagataella phaffii |
| BBa_K3841049 | DNA | Homology arm for neutral insertion site 2 in Komagataella phaffii |
| BBa_K3841050 | DNA | Homology arm for neutral insertion site 2 in Komagataella phaffii |
| BBa_K3841051 | DNA | Homology arm for neutral insertion site 2 in Komagataella phaffii |
| BBa_K3841052 | DNA | Homology arm for neutral insertion site 2 in Komagataella phaffii |
| BBa_K3841053 | DNA | Homology arm for neutral insertion site 3 in Komagataella phaffii |
| BBa_K3841054 | DNA | Homology arm for neutral insertion site 3 in Komagataella phaffii |
| BBa_K3841055 | DNA | Homology arm for neutral insertion site 3 in Komagataella phaffii |
| BBa_K3841056 | DNA | Homology arm for neutral insertion site 3 in Komagataella phaffii |
| BBa_K3841057 | DNA | Homology arm for neutral insertion site 3 in Komagataella phaffii |
| BBa_K3841058 | DNA | Homology arm for neutral insertion site 3 in Komagataella phaffii |
| BBa_K3841059 | DNA | Homology arm for neutral insertion site 3 in Komagataella phaffii |
| BBa_K3841060 | DNA | Homology arm for neutral insertion site 3 in Komagataella phaffii |
| BBa_K3841061 | DNA | Homology arm for neutral insertion site 4 in Komagataella phaffii |
| BBa_K3841062 | DNA | Homology arm for neutral insertion site 4 in Komagataella phaffii |
| BBa_K3841063 | DNA | Homology arm for neutral insertion site 4 in Komagataella phaffii |
| BBa_K3841064 | DNA | Homology arm for neutral insertion site 4 in Komagataella phaffii |
| BBa_K3841065 | DNA | Homology arm for neutral insertion site 4 in Komagataella phaffii |
| BBa_K3841066 | DNA | Homology arm for neutral insertion site 4 in Komagataella phaffii |
| BBa_K3841067 | DNA | Homology arm for neutral insertion site 4 in Komagataella phaffii |
| BBa_K3841068 | DNA | Homology arm for neutral insertion site 4 in Komagataella phaffii |
gRNA Backbone
| Part name | Part Type | Description |
|---|---|---|
| BBa_K3841003 | DNA | gRNA backbone used for Cas9 mediated double-stranded breaks. |
Reporter Collection for Komagataella phaffii
The repertoire of reporters validated for K. phaffii GS115 in the iGEM registry is limited. This reporter collection aimed to expand on reporters to be used in this yeast strain. We designed several methanol-dependant expression cassettes containing various fluorescent reporter proteins, all yeast optimized: GFP, mOrange (RFP) and Venus (YFP). Further, we provided a Venus version which has been codon-optimized for K. phaffii. We also added Soy Leghemoglobin, a red protein due to its heme-group. The expression cassettes are ideal for quantifying promoter strength.
Fluorescent Proteins
| Part name | Part Type | Description |
|---|---|---|
| BBa_K3841006 | Reporter | Venus Yellow Fluorescent Protein for Komagataella phaffii |
| BBa_K3841022 | Reporter | Expression cassette of yeast optimized Venus from methanol-dependent promoter AOX1 |
| BBa_K3841024 | Reporter | Expression cassette of yeast optimized Venus from methanol-dependent promoter |
| BBa_K3841025 | Reporter | Expression cassette of yeast optimized Venus from methanol-dependent promoter |
| BBa_K3841026 | Reporter | Expression cassette of yeast optimized Venus from regulated FLD1 promoter |
| BBa_K3841092 | Reporter | Expression cassette of GFP from methanol-dependent promoter |
| BBa_K3841093 | Reporter | Expression cassette of mOrange from methanol-dependent promoter |
| BBa_K3841094 | Reporter | Expression cassette of yeast optimized Venus from methanol-dependent promoter |
Soy Leghemoglobin - A Red Protein
| Part name | Part Type | Description |
|---|---|---|
| BBa_K3841014 | CDS | Soy Leghemoglobin |
| BBa_K3841088 | Reporter | Expression cassette of soy leghemoglobin from methanol-dependent promoter |
| BBa_K3841089 | Reporter | Expression cassette of soy leghemoglobin from methanol-dependent promoter |
| BBa_K3841090 | Reporter | Expression cassette of soy leghemoglobin from methanol-dependent promoter |
| BBa_K3841091 | Reporter | Expression cassette of soy leghemoglobin from regulated promoter |
Vector Backbone
| Part name | Part Type | Description |
|---|---|---|
| BBa_K3841023 | Plasmid_backbone | pGGAselect destination plasmid for Golden Gate Assembly desgined by New England Biolabs (NEB) |
Methanotropic Yeast Toolbox for Komagataella phaffii
K. phaffii GS115 is used as an industrial workhorse for heterologous protein production. It can grow on methanol but does not consume methane. It only requires one enzymatic step to convert methane to methanol, thus creating a powerful methane-consuming microbial cell-factory. With our newly developed genetic toolbox for engineering K. phaffi, we wanted to test our neutral integration sites. To integrate the necessary parts for methanotrophy, we designed a repertoire of repair templates encoding the pMMO subunits from Methylococcus capsulatus: PmoA, PmoB and PmoC.
Integration of pMMO for Methane Valorization
| Part Name | Part Type | Description |
|---|---|---|
| BBa_K3841009 | CDS | Particulate methane monooxygenase beta subunit (PmoA) |
| BBa_K3841010 | CDS | Particulate methane monooxygenase alpha subunit (PmoB) |
| BBa_K3841011 | CDS | Particulate methane monooxygenase gamma subunit (PmoC) |
| BBa_K3841015 | CDS | Particulate methane monooxygenase alpha subunit (PmoB) fused to Venus |
| BBa_K3841036 | Tag | Plasma membrane signal peptide for Komagataella phaffii |
| BBa_K3841069 | Composite | Expression cassette of pMMO beta subunit (PmoA) with constitutive promoter |
| BBa_K3841070 | Composite | Expression cassette of pMMO beta subunit (PmoA) with constitutive promoter and signal peptide |
| BBa_K3841071 | Composite | Expression cassette of pMMO alpha subunit (PmoB) with constitutive promoter |
| BBa_K3841072 | Composite | Expression cassette of pMMO alpha subunit (PmoB) with constitutive promoter and signal peptide |
| BBa_K3841073 | Composite | Expression cassette of pMMO alpha subunit (PmoB) fused to Venus tag with constitutive promoter |
| BBa_K3841074 | Composite | Expression cassette of pMMO alpha subunit (PmoB)fused to Venus tag with constitutive promoter and signal peptide |
| BBa_K3841075 | Composite | Expression cassette of pMMO gamma subunit (PmoC) with constitutive promoter |
| BBa_K3841076 | Composite | Expression cassette of pMMO gamma subunit (PmoC) with constitutive promoter and signal peptide |
Promoters
| Part Name | Part Type | Description |
|---|---|---|
| BBa_K3841000 | Promoter | GAP1 promoter |
| BBa_K3841012 | Promoter | TEF1 promoter |
| BBa_K3841001 | Promoter | Methanol regulated promoter AOX1 |
| BBa_K3841007 | Promoter | Methanol regulated promoter AOX1 |
| BBa_K3841008 | Promoter | Methanol regulated promoter AOX1 |
| BBa_K3841005 | Promoter | FLD1 promoter |
| BBa_K3841013 | Terminator | TEF1 terminator |
Vector Backbone
| Part Name | Part Type | Description |
|---|---|---|
| BBa_K3841023 | Plasmid_backbone | pGGAselect destination plasmid for Golden Gate Assembly desgined by New England Biolabs (NEB) |
