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 (Link opens in a new window) Article (Link opens in a new window).
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 (Link opens in a new window)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 (Link opens in a new window) Article (Link opens in a new window).
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 (Link opens in a new window) Article (Link opens in a new window).
Diatom uLoop Assembly Kit - #1000000238
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
- Inventory
Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key
| Kanamycin | |
| Spectinomycin |
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 |