Jul 29, 2022
Golden Gate Assembly_Perkinsus
- Ross F. Waller1,
- Elin Einarsson1,
- Imen Lassadi1
- 1University of Cambridge
- Protist Research to Optimize Tools in Genetics (PROT-G)
- Perkinsus
Protocol Citation: Ross F. Waller, Elin Einarsson, Imen Lassadi 2022. Golden Gate Assembly_Perkinsus. protocols.io https://dx.doi.org/10.17504/protocols.io.bv3zn8p6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 25, 2021
Last Modified: July 29, 2022
Protocol Integer ID: 51033
Keywords: aquatic parasite lineage, modular golden gate plasmid assembly system for expression, experimental genetic tool, few tools for transgene expression, perkinsus as an aquatic parasite, golden gate assembly-perkinsus the phylum perkinsozoa, genetic transformation, power of experimental genetic tool, plasmid assembly system, multiple gene, excellent genetic model, aquatic parasite, gene, phylum perkinsozoa, peptide, dinoflagellate, viral 2a peptide, parasites of algae, new promoters of variable expression strength, golden gate assembly-perkinsus, transgene expression, perkinsus spp, parasite, natural mollusc population
Funders Acknowledgements:
Gordon and Betty Moore Foundation
Grant ID: 4977
Gordon and Betty Moore Foundation
Grant ID: 4977.1
Abstract
The phylum Perkinsozoa is an aquatic parasite lineage that has devastating effects on commercial and natural mollusc populations, and also comprises parasites of algae, fish and amphibians. They are related to dinoflagellates and apicomplexans and thus offer excellent genetic models for both parasitological and evolutionary studies. Genetic transformation was previously achieved for Perkinsus spp. but with few tools for transgene expression and limited selection efficacy. We have expanded the power of experimental genetic tools for Perkinsus using P. marinus as a model. We constructed a modular Golden Gate plasmid assembly system for expression of multiple genes simultaneously as the basis of this effort. This has provided a versatile platform enabling several further developments: efficient selection systems for three drugs, puromycin, bleomycin and blasticidin; eleven new promoters of variable expression strengths; and bi-cistronic transcripts using the viral 2A peptides can couple selection to the maintenance of the expression of a transgene of interest. Collectively, thesenew tools provide great new capacity to genetically modify and study Perkinsus as an aquatic parasite and evolutionary model.
Attachments
Guidelines
This protocol assumes that
- All modules being assembled have been “domesticated” (are free of internalBsaI andBpiI recognition sequences)
- All module junctions being assembled have a unique set of compatible overhangs that specifies their order of assembly
- The acceptor plasmid has a different antibiotic resistant gene to that of all of the modules plasmids being assembled into it
It has been found that the isoschizomer BbsI (supplied NEB) loses activity very quickly and has a lot of star-activity so it is advised not to use this.
Protocol materials
BpiI (BbsI) (10 U/µL)Thermo FisherCatalog #ER1011
Buffer G (10X)Thermo FisherCatalog #BG5
T4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S
CutSmart® BufferNew England BiolabsCatalog #B7204S
BsaI - 5,000 unitsNew England BiolabsCatalog #R0535L
Troubleshooting
Golden Gate cloning into Level 14 steps
To assemble fragments in Level 1 acceptors using BsaI, add all of the following to a PCR tube and make the reaction volume up to 20 µL with sterile distilled water.
- 100 ng to 200 ng of acceptor plasmid
- Plasmids containing each module/part to be inserted. Use a 2:1 molar ratio of insert:acceptor. NB: If your sequence is ~1KB or less, it can be added directly to this assembly reaction as a linear PCR amplicon.
- 20 units BsaI (1 µL of BsaI - 5,000 unitsNew England BiolabsCatalog #R0535L )
- 2 µL CutSmart® BufferNew England BiolabsCatalog #B7204S
- 400 units T4 DNA Ligase (1 µL of T4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S )
- 2 µL of 10 millimolar (mM) ATP
Incubate your reaction in a thermocycler for 3 cycles of 00:10:00 40 °C , 00:10:00 16 °C , followed by a single cycle of 00:10:00 50 °C , 00:20:00 80 °C , 4 °C forever.
50m
Use 5 µL of this one-pot digestion-ligation reaction product to transform E.coli cells.
Positive clones can be selected using LB agar with an antibiotic appropriate to the backbone of the acceptor vector. If your acceptor vector contained the lacZ cassette, include 0.5 millimolar (mM) IPTG and 0.04 mg/mL and X-gal. Select 3 -10 (white) colonies for restriction analysis and confirmation by sequencing.