Team:DTU-Denmark/Parts

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)