Diatom uLoop Assembly Kit
(Kit # 1000000238 )

Depositing Lab:   Christopher Dupont

This collection includes a standard set of universal loop (uLoop) backbones, fluorescent proteins, protein tags, and signal peptides for localization. The collection also contains a selection of diatom promoters and terminators from the organism Phaeodactylum tricornutum on vectors compatible with the uLoop cloning system.

This kit will be sent as bacterial glycerol stocks in 96-well plate format.

Original Publication

Universal loop assembly: open, efficient and cross-kingdom DNA fabrication. Pollak B, Matute T, Nunez I, Cerda A, Lopez C, Vargas V, Kan A, Bielinski V, von Dassow P, Dupont CL, Federici F. Synth Biol (Oxf). 2020;5(1):ysaa001. doi: 10.1093/synbio/ysaa001. Epub 2020 Feb 5. PubMed Article .

Description

Compatible with the uLoop cloning system, this collection of plasmids contains all the parts necessary (oriT, ShBle, CenArsHis) to build conjugatable episomes for expression in diatoms. The plasmid backbones have so far been shown to be successfully maintained in Phaeodactylum tricornutum and Thalassiosira pseudonana. This kit also contains a selection of verified promoters and terminators from P. tricornutum with expression levels spanning four orders of magnitude, allowing for fine-tuned control of gene expression.

Users can easily build their own level 0 parts to add to their uLoop library by using the pL0 backbone. Genes encoding for proteins of interest could then be made into CD or DE parts, for example, and then tagged using a variety of fluorescent proteins or signal peptides for localizing proteins to the nucleus, endoplasmic reticulum, peroxisome, mitochondria, chloroplast, pyrenoid compartment, or extracellularly. Genes can also be expressed in a bicistronic manner using the provided 2A peptide. Using the power of Loop assembly and the parts contained in this kit, individual transcription units can be built and combined to quickly and easily construct large biosynthetic pathways for expression in diatoms.

• Figure 1. Universality of uLoop. (A) Decoupling of Loop assembly logic. uLoop decouples the propagation elements specific to the organism of choice from the universal assembly logic inbuilt in Loop vectors. (B) Creation of uLoop species-specific vectors from Loop. In uLoop, propagation elements required for transfer and maintenance of plasmids in target organisms are used as modular L0 parts. During the assembly routine, these cassettes are assembled into plasmids alongside other transcription units to generate species-specific plasmids capable of transfer and propagation in the organism of interest. (C) Overhangs and restriction sites used in uLoop/Loop assembly. BsaI overhangs follow the common syntax, and SapI overhangs are the same as those described in Loop assembly, α(ATG), β(GCA), γ(TAC), ε(CAG) and ω(GGT). (D) Source of uLoop vector kits. Vectors from the synthetic biology community and the pCAMBIA vector were domesticated for BsaI and SapI and ‘minimised’ by removing elements not related to basic plasmid function. Two antibiotic resistance versions were generated and then the Loop schema was incorporated to generate odd and even plasmids for each version of the vector kits.

Kit Documentation

Protocol for how to perform Loop Assembly using the plasmids in this kit.

Second publication (containing the expression data for the promotors and terminators included in the kit):
Validating a Promoter Library for Application in Plasmid-Based Diatom Genetic Engineering. Garza EA, Bielinski VA, Espinoza JL, Orlandi K, Alfaro JR, Bolt TM, Beeri K, Weyman PD, Dupont CL. ACS Synth Biol. 2023 Nov 17;12(11):3215-3228. doi: 10.1021/acssynbio.3c00163. PubMed Article .

How to Cite this Kit

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.

For your Materials and Methods section:

“The Diatom uLoop Assembly Kit was a gift from Christopher Dupont (Addgene kit #1000000238).”

For your Reference section:

Universal loop assembly: open, efficient and cross-kingdom DNA fabrication. Pollak B, Matute T, Nunez I, Cerda A, Lopez C, Vargas V, Kan A, Bielinski V, von Dassow P, Dupont CL, Federici F. Synth Biol (Oxf). 2020;5(1):ysaa001. doi: 10.1093/synbio/ysaa001. Epub 2020 Feb 5. PubMed Article .

Diatom uLoop Assembly Kit - #1000000238

Inventory

Well	Plasmid	Resistance
A / 1	pCA_o-1	Kanamycin
A / 2	pCA_o-2	Kanamycin
A / 3	pCA_o-3	Kanamycin
A / 4	pCA_o-4	Kanamycin
A / 5	pL0R_lacZ	Spectinomycin
A / 6	pCA_e-1	Spectinomycin
A / 7	pCA_e-2	Spectinomycin
A / 8	pCA_e-3	Spectinomycin
A / 9	pCA_e-4	Spectinomycin
A / 10	pL0_AB-H4	Spectinomycin
A / 11	pL0_AB-NR	Spectinomycin
A / 12	pL0_AB-P49	Spectinomycin
B / 1	pL0_AB-OEE3	Spectinomycin
B / 2	pL0_AC-epyC	Spectinomycin
B / 3	pL0_AC-BCA	Spectinomycin
B / 4	pL0_AC-47740	Spectinomycin
B / 5	pL0_AC-GEF	Spectinomycin
B / 6	pL0_AC-51183	Spectinomycin
B / 7	pL0_AC-54730	Spectinomycin
B / 8	pL0_AC-27877	Spectinomycin
B / 9	pL0_AC-fbaC5	Spectinomycin
B / 10	pL0_AC-CaMK2	Spectinomycin
B / 11	pL0_AC-act1	Spectinomycin
B / 12	pL0_AC-sit4	Spectinomycin
C / 1	pL0_AC-NRT	Spectinomycin
C / 2	pL0_AC-HO	Spectinomycin
C / 3	pL0_AC-lysA	Spectinomycin
C / 4	pL0_AC-vapol	Spectinomycin
C / 5	pL0_AC-fcpF	Spectinomycin
C / 6	pL0_AC-P49	Spectinomycin
C / 7	pL0_AC-fcpB	Spectinomycin
C / 8	pL0_AC-H4	Spectinomycin
C / 9	pL0_AC-NR	Spectinomycin
C / 10	pL0_AC-GPI1	Spectinomycin
C / 11	pL0_AC-Fld	Spectinomycin
C / 12	pL0_AC-dph1	Spectinomycin
D / 1	pL0_AC-glna	Spectinomycin
D / 2	pL0_AC-acc2	Spectinomycin
D / 3	pL0_AC-CEN_ARS_HIS	Spectinomycin
D / 4	pL0_AF-spacer	Spectinomycin
D / 5	pL0_BC-chl_sig_pep	Spectinomycin
D / 6	pL0_BC-pyr_comp_sig_pep	Spectinomycin
D / 7	pL0_BC-sec_sig_pep	Spectinomycin
D / 8	pL0_BC-mito_sig_pep	Spectinomycin
D / 9	pL0_BC-nuc_sig_pep	Spectinomycin
D / 10	pL0_BC-ER_sig_pep	Spectinomycin
D / 11	pL0_CD-GUS	Spectinomycin
D / 12	pL0_CD-PT2A	Spectinomycin
E / 1	pL0_CD-eYFP	Spectinomycin
E / 2	pL0_CD-roGFP	Spectinomycin
E / 3	pL0_CD-greenlantern	Spectinomycin
E / 4	pL0_CD-eCFP	Spectinomycin
E / 5	pL0_CD-mcherry	Spectinomycin
E / 6	pL0_CD-eYGFP-uv	Spectinomycin
E / 7	pL0_CD-mRFP1	Spectinomycin
E / 8	pL0_CD-mTurquoise2	Spectinomycin
E / 9	pL0_CD-sfGFP	Spectinomycin
E / 10	pL0_CD-Venus	Spectinomycin
E / 11	pL0_CD-eGFP	Spectinomycin
E / 12	pL0_CE-OriT	Spectinomycin
F / 1	pL0_DE-3xStop	Spectinomycin
F / 2	pL0_DE-Per_sig_pep	Spectinomycin
F / 3	pL0_DE-mRuby3	Spectinomycin
F / 4	pL0_DE-RFP1	Spectinomycin
F / 5	pL0_DE_mTurquoise2	Spectinomycin
F / 6	pL0_DE-sfGFP	Spectinomycin
F / 7	pL0_DE-Venus	Spectinomycin
F / 8	pL0_EF-BCA	Spectinomycin
F / 9	pL0_EF-47740	Spectinomycin
F / 10	pL0_EF-GEF	Spectinomycin
F / 11	pL0_EF-51183	Spectinomycin
F / 12	pL0_EF-27877	Spectinomycin
G / 1	pL0-EF-54730	Spectinomycin
G / 2	pL0_EF-fcpA	Spectinomycin
G / 3	pL0_EF-OEE3	Spectinomycin
G / 4	pL0_EF-fbaC5	Spectinomycin
G / 5	pL0_EF-CaMK2	Spectinomycin
G / 6	pL0_EF-sit4	Spectinomycin
G / 7	pL0_EF-NRT	Spectinomycin
G / 8	pL0_EF-HO	Spectinomycin
G / 9	pL0_EF-act1_tr	Spectinomycin
G / 10	pL0_EF-act1_full	Spectinomycin
G / 11	pL0_EF-lysA	Spectinomycin
G / 12	pL0_EF-vapol	Spectinomycin
H / 1	pL0_EF-GPI1	Spectinomycin
H / 2	pL0_EF-P49	Spectinomycin
H / 3	pL0_EF-fcpB	Spectinomycin
H / 4	pL0_EF-H4	Spectinomycin
H / 5	pL0_EF-NR	Spectinomycin
H / 6	pL0_EF-PtBle	Spectinomycin
H / 7	pL0_EF-Fld	Spectinomycin
H / 8	pL0_EF-dph1	Spectinomycin
H / 9	pL0_EF-glna	Spectinomycin
H / 10	pL0_EF-acc2	Spectinomycin
H / 11	pL0_EF-epyC	Spectinomycin

Data calculated @ 2024-11-21