TITLE: Golden Gate Assembly_Perkinsus
AUTHORS: Ross   F. Waller, Elin  Einarsson, Imen   Lassadi

[DESCRIPTION]
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.

[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.

[STEPS]
1. 
3. Incubate your reaction in a thermocycler for 3 cycles of  10 min 40 °C ,  10 min16 °C, followed by a single cycle of 10 min 50 °C , 20 min80 °C,  4 °C forever. 
4. Use 5 µLof this one-pot digestion-ligation reaction product to transform E.coli cells.
5. 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/mLand X-gal. Select 3 -10 (white) colonies for restriction analysis and confirmation by sequencing.

2. To assemble fragments in Level 0 and level 2 acceptors using BpiI, add all of the following to a PCR tube and make the reaction volume up to 20 µLl with sterile distilled water.
100 ngto 200 ngof 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 amplicon
10 unitsBpiI (1 µLof  )
2 µL  
400 units T4 DNA Ligase (1 µLof   )
2 µLof 10 millimolar (mM) ATP


3. Incubate your reaction in a thermocycler for 3 cycles of  10 min37 °C, 10 min16 °C, followed by a single cycle of 10 min37 °C, 20 min65 °C, 4 °C forever. 
4. Use 5 µLof this one-pot digestion-ligation reaction product to transform E.coli cells.
5. 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/mLX-gal. Select 3 -10 (white) colonies for restriction analysis and confirmation by sequencing.

2. 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 unitsBsaI (1 µLof  )
2 µL  
400 units T4 DNA Ligase (1 µLof   )
2 µLof 10 millimolar (mM)ATP