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Nuclear Transformation of Chlamydomonas reinhardtii by Electroporation
Published: May 5, 2018 DOI: 10.21769/BioProtoc.2837 Views: 9767
Edited by: Adam Idoine Reviewed by: Ru Zhang
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Abstract
The unicellular green alga Chlamydomonas reinhardtii is an important model organism for studying photosynthesis, acclimation to abiotic stress, cilia biology, and many other biological processes. Many molecular biology tools exist for interrogating gene function including the ability to easily transform the nuclear genome of Chlamydomonas. While technical advances such as TALENs, ZFNs and CRISPR are making it easier to precisely edit the nuclear genome, the efficiency of such methods in Chlamydomonas is at present very low. In contrast, random insertion by nuclear transformation tends to be a much more efficient process. This protocol describes a method for transformation of the Chlamydomonas nuclear genome by electroporation. The protocol requires at least 3 days of work and generally results in the appearance of small colonies within 1-2 weeks.
Keywords: Algae
Nuclear transformation
Electroporation
Fluorescent fusion protein
Venus
mCherry
FLAG tag
His tag
Paromomycin
Hygromycin
Background
Numerous molecular, genetic and genomic resources make Chlamydomonas reinhardtii (Chlamydomonas hereafter) an excellent model organism for studies on diverse biological processes. Many techniques have been developed to transform the Chlamydomonas nucleus, chloroplast and mitochondria including particle bombardment (Boynton et al., 1988), glass bead transformation (Kindle, 1990), and electroporation (Shimogawara et al., 1998). Nuclear mutants may be generated by exposure of Chlamydomonas cells to physical or chemical mutagens (e.g., UV light or ethyl methanesulfonate), but are often obtained by random insertional mutagenesis of transgenic DNA. Since the efficiency of homologous recombination for nuclear transformation in Chlamydomonas is very low (Zorin et al., 2009; Jinkerson and Jonikas, 2015), transformed DNA is generally integrated into the nuclear genome at random sites. A number of techniques exist for subsequently identifying the insertion sites of the ectopic DNA including classical genetic mapping (Rymarquis et al., 2005), TAIL-PCR (Dent et al., 2005), and next-generation sequencing of individual mutants (Dutcher et al., 2012) or large mutant libraries (Zhang et al., 2014; Li et al., 2016). While recent technical advances have led to improvements in targeted genome editing in Chlamydomonas using CRISPR/Cas9 (Baek et al., 2016; Shin et al., 2016; Ferenczi et al., 2017; Greiner et al., 2017) and zinc-finger nucleases (Sizova et al., 2013; Greiner et al., 2017), random insertional mutagenesis is still a preferred method to generate mutant libraries for forward and reverse genetics.
This protocol describes a detailed method for nuclear transformation of Chlamydomonas by electroporation. It can be used to generate random insertion mutants using a plasmid fragment conferring antibiotic resistance (Jinkerson and Jonikas, 2015) or for the expression of fluorescent fusion proteins using well-established, publically-available expression vectors. Once a suitable DNA fragment has been obtained or generated, the transformation protocol takes two days and generally results in visible, isolated colonies within 1-2 weeks.
Materials and Reagents
- Pipette tips with filters
- Sterile 0.6 ml microcentrifuge tubes
- Sterile 15 ml centrifuge tubes
- Sterile 50 ml centrifuge tubes
- 0.4 cm gap electroporation cuvettes (Bio-Rad Laboratories, catalog number: 1652088 )
- Petri dishes
- Blue Sharpie (permanent) marker pen
- Sterile plastic inoculating loops (VWR, catalog number: 12000-810 )
- Parafilm
- Optional: plasmid pLM005 (available from the Chlamydomonas Resource Center, www.chlamy.org)
- Optional: plasmid pLM006 (available from the Chlamydomonas Resource Center, www.chlamy.org)
- Optional: plasmid pMO449 (available from the Chlamydomonas Resource Center, www.chlamy.org)
- Optional: wild-type strain CC-1690 21gr+ (available from the Chlamydomonas Resource Center, www.chlamy.org)
- 70% ethanol
- Canned air (Fisher Scientific, catalog number: 23-022-523 )
- Tris base (Biopioneer, catalog number: C0060 )
- Hydrochloric acid (HCl) (Fisher Scientific, catalog number: A144-212 )
- Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P0662 )
- Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
- Ammonium chloride (NH4Cl) (Sigma-Aldrich, catalog number: A4514 )
- Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C3306 )
- Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 230391 )
- Ethylenediaminetetraacetic acid, disodium salt, dihydrate (EDTA·Na2·2H2O) (Sigma-Aldrich, catalog number: E5134 )
- Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 221473 )
- Ammonium molybdate tetrahydrate [(NH4)6Mo7O24·7H2O] (Sigma-Aldrich, catalog number: A7302 )
- Sodium selenite (Na2SeO3) (Sigma-Aldrich, catalog number: S5261 )
- Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z4750 )
- Manganese(II) chloride tetrahydrate (MnCl2·4H2O) (Sigma-Aldrich, catalog number: M3634 )
- Iron(III) chloride hexahydrate (FeCl3·6H2O) (Sigma-Aldrich, catalog number: F2877 )
- Copper(II) chloride dihydrate (CuCl2·2H2O) (Sigma-Aldrich, catalog number: C3279 )
- Glacial acetic acid (Fisher Scientific, catalog number: A38-212 )
- Sodium carbonate (Na2CO3) (Sigma-Aldrich, catalog number: S7795 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
- Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L5750 )
- Agar (Caisson Laboratories, catalog number: A038 )
- Paromomycin sulfate salt (Paro) (Sigma-Aldrich, catalog number: P8692 )
- Hygromycin B (Hyg) 50 mg/ml solution (Clontech, catalog number: 631309 )
- Stock solutions (see Recipes)
- 1 M Tris Base (1 L, 50x)
- Solution A for TAP (500 ml, 100x)
- 125 mM EDTA·Na2 pH 8.0 (300 ml)
- 285 µM (NH4)6Mo7O24 (250 ml)
- 1 mM Na2SeO3 (250 ml)
- 100 mg/ml paromomycin (1 ml)
- 1 M Tris Base (1 L, 50x)
- Micronutrient stock solutions (see Recipes)
- 25 mM EDTA·Na2 (250 ml)
- 28.5 µM (NH4)6Mo7O24 (250 ml)
- 0.1 mM Na2SeO3 (250 ml)
- Zn-EDTA (250 ml)
- Mn-EDTA (250 ml)
- Fe-EDTA (250 ml)
- Cu-EDTA (250 ml)
- 25 mM EDTA·Na2 (250 ml)
- TAP liquid medium (see Recipes)
- TAP 40 mM sucrose (see Recipes)
- TAP + 20 µg/ml Paro selective agar medium (see Recipes)
- TAP + 25 µg/ml Hyg selective agar medium (see Recipes)
*Note: Materials used for generating plasmids of interest (polymerases, restriction enzymes, ligases, buffers, etc.) are not described here.
Equipment
- Sterile 250 ml culture flasks (e.g., Corning, catalog number: 70980-250 )
- Sterile 1 or 2 L culture flasks (e.g., DWK Life Sciences, Kimble®, catalog numbers: 26500-1000 or 26500-2000 )
- Pipettes
- Centrifuge (e.g., Eppendorf, model: 5810 R ) and microcentrifuge (e.g., Eppendorf, model: 5424 )
- NanoDrop 2000 (or similar) spectrophotometer
- Hemacytometer (e.g., Sigma-Aldrich, catalog number: Z359629 ) or automated cell counter (e.g., Bio-Rad Laboratories, model: TC20 )
- Water bath with thermometer
- Biosafety cabinet (with optional UV light)
Note: A laminar flow hood can also be used - Gene Pulser XCell Electroporator (Bio-Rad Laboratories, catalog number: 1652660 )
- Tube shaker (e.g., Thermo Fisher Scientific, catalog number: T415110Q )
Procedure
Category
Plant Science > Phycology > DNA
Plant Science > Phycology > Nuclear transformation
Molecular Biology > DNA > Transformation
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