Jul 28, 2022

Public workspaceGolden Gate Assembly_Perkinsus

DOI
dx.doi.org/10.17504/protocols.io.bv3zn8p6
  • 1University of Cambridge
  • Protist Research to Optimize Tools in Genetics (PROT-G)
  • Perkinsus
Sara Chelaghma
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DOI: dx.doi.org/10.17504/protocols.io.bv3zn8p6
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 24, 2021
Last Modified: July 28, 2022
Protocol Integer ID: 51033
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
Icon for GoldenGate_plasmids.xlsx
GoldenGate_plasmids....
37KB
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
ReagentBpiI (BbsI) (10 U/µL)Thermo FisherCatalog #ER1011
ReagentBuffer G (10X)Thermo FisherCatalog #BG5
ReagentT4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S
ReagentCutSmart® BufferNew England BiolabsCatalog #B7204S
ReagentBsaI - 5,000 unitsNew England BiolabsCatalog #R0535L
ReagentT4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S

Step case

Golden Gate cloning into Level 1
4 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 Amount20 µL with sterile distilled water.
  • Amount100 ng to Amount200 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.
  • Amount20 units BsaI (Amount1 µL of ReagentBsaI - 5,000 unitsNew England BiolabsCatalog #R0535L )
  • Amount2 µL ReagentCutSmart® BufferNew England BiolabsCatalog #B7204S
  • Amount400 units T4 DNA Ligase (Amount1 µL of ReagentT4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S )
  • Amount2 µL of Concentration10 millimolar (mM) ATP

Incubate your reaction in a thermocycler for 3 cycles of Duration00:10:00 Temperature40 °C , Duration00:10:00 Temperature16 °C , followed by a single cycle of Duration00:10:00 Temperature50 °C , Duration00:20:00 Temperature80 °C , Temperature4 °C forever.
50m
Use Amount5 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 Concentration0.5 millimolar (mM) IPTG and Concentration0.04 mg/mL and X-gal. Select 3 -10 (white) colonies for restriction analysis and confirmation by sequencing.