We thank N. Dean for providing the original mCherry FP sequence, M. Gerami-Nejad for construction of plasmids, B. Larson for technical assistance, and T. Heisel for helpful advice during the development of this project. J.B. was supported by the European Research Council Advanced Award 340087 (RAPLODAPT). Microscopy and imaging systems were provided by the University of Minnesota Pediatrics Foundation and the University of Minnesota Imaging Center.

1. Isolate Template Plasmids from E. coli
<ol>
	<li>Grow E. coli containing the template plasmid overnight in 10 ml lysogeny broth (LB) + 200 mg/L ampicillin (AMP) at 37 &#176;C with shaking.</li>
	<li>Harvest cells by centrifuging at 6,000 x g for 2 min.</li>
	<li>Decant liquid, isolate and purify DNA from E. coli cells by a standard method as described previously in Ausubel et al.8.</li>
	<li>Resuspend DNA in Tris-EDTA (TE; 10 mM Tris, pH 8.0, 1 mM EDTA, pH ...

<ol>
	<li>Bendel, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonization+and+epithelial+adhesion+in+the+pathogenesis+of+neonatal+candidiasis.">Colonization and epithelial adhesion in the pathogenesis of neonatal candidiasis.</a> Semin. Perinatol. 27 (5), 357-364 (2003).</li><li>Gerami-Nejad, M., Berman, J., Gale, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cassettes+for+PCR-mediated+construction+of+green,+yellow,+and+cyan+fluorescent+protein+fusions+in+Candida+albicans.">Cassettes for PCR-mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans.</a> Yeast. 18 (9), 859-864 (2001).</li><li>Gerami-Nejad, M., Dulmage, K., Berman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additional+cassettes+for+epitope+and+fluorescent+fusion+proteins+in+Candida+albicans.">Additional cassettes for epitope and fluorescent fusion proteins in Candida albicans.</a> Yeast. 26 (7), 399-406 (2009).</li><li>Gerami-Nejad, M., Forche, A., McClellan, M., Berman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+protein+function+in+clinical+C.+albicans+isolates.">Analysis of protein function in clinical C. albicans isolates.</a> Yeast. 29 (8), 303-309 (2012).</li><li>Gerami-Nejad, M., Hausauer, D., McClellan, M., Berman, J., Gale, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cassettes+for+the+PCR-mediated+construction+of+regulatable+alleles+in+Candida+albicans.">Cassettes for the PCR-mediated construction of regulatable alleles in Candida albicans.</a> Yeast. 21 (5), 429-436 (2004).</li><li>Gonia, S., Larson, B., Gale, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR-mediated+gene+modification+strategy+for+construction+of+fluorescent+protein+fusions+in+Candida+parapsilosis.">PCR-mediated gene modification strategy for construction of fluorescent protein fusions in Candida parapsilosis.</a> Yeast. 33 (2), 63-69 (2016).</li><li>Milne, S. W., Cheetham, J., Lloyd, D., Aves, S., Bates, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cassettes+for+PCR-+mediated+gene+tagging+in+Candida+albicans+utilizing+nourseothricin+resistance.">Cassettes for PCR- mediated gene tagging in Candida albicans utilizing nourseothricin resistance.</a> Yeast. 28 (12), 833-841 (2011).</li><li>Ausubel, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Molecular Biology. , (1995).</li><li>Wilson, R. B., Davis, D., Mitchell, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+hypothesis+testing+with+Candida+albicans+through+gene+disruption+with+short+homology+regions.">Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions.</a> J. Bacteriol. 181 (6), 1868-1874 (1999).</li><li>Pulver, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rsr1+focuses+Cdc42+activity+at+hyphal+tips+and+promotes+maintenance+of+hyphal+development+in+Candida+albicans.">Rsr1 focuses Cdc42 activity at hyphal tips and promotes maintenance of hyphal development in Candida albicans.</a> Eukaryotic Cell. 12 (4), 482-495 (2013).</li><li>Falgier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+species+differ+in+their+interactions+with+immature+human+gastrointestinal+epithelial+cells.">Candida species differ in their interactions with immature human gastrointestinal epithelial cells.</a> Pediatr. Res. 69 (5), 384-389 (2011).</li><li>Nosek, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+the+pathogenic+yeast+Candida+parapsilosis.">Genetic manipulation of the pathogenic yeast Candida parapsilosis.</a> Curr. Genet. 42 (1), 27-35 (2002).</li><li>Zemanova, J., Nosek, J., Tomaska, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+transformation+of+the+pathogenic+yeast+Candida+parapsilosis.">High-efficiency transformation of the pathogenic yeast Candida parapsilosis.</a> Curr. Genet. 45 (3), 183-186 (2004).</li><li>Benjamin, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+candidiasis+among+extremely+low+birth+weight+infants:+risk+factors,+mortality+rates,+and+neurodevelopmental+outcomes+at+18+to+22+months.">Neonatal candidiasis among extremely low birth weight infants: risk factors, mortality rates, and neurodevelopmental outcomes at 18 to 22 months.</a> Pediatrics. 117 (1), 84-92 (2006).</li><li>Kullberg, B. J., Arendrup, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+Candidiasis.">Invasive Candidiasis.</a> N Engl J Med. 373 (15), 1445-1456 (2015).</li></ol>

Construction of epitope tagged sequences in Candida species using the PCR-mediated gene modification strategy described above can be summarized as a three-step process. First, a cassette is made by PCR that encodes both the sequence desired for integration and regions homologous to the locus of insertion into the yeast genome. Second, the yeast cells to be transformed are made chemically competent with lithium acetate and co-incubated with the cassette. Third, the cells are plated on selective media to recover t...

Candida species are commensal fungi that colonize the intestinal and genitourinary tracts of all humans. Under conditions of immunodeficiency, such as that occur with premature birth or immunosuppressive effects from treatments for cancer, Candida species can become opportunistic pathogens. Of the Candida species, Candida albicans is the most prevalent fungal colonizer and causes the majority of invasive fungal infections. Other Candida species such as C. glabrata, C. parapsilosis, C. tropicalis, and C. kruseii also cause serious infections in immunocompromised patients, with some exhibiting intrinsic resistance to commonly used anti-fungal antibiotics such as fluconazole and amphotericin B. Hence, infections with some of these species are being observed more frequently, especially in patients being treated prophylactically with anti-fungal agents. Even with appropriate and timely anti-fungal treatment, invasive Candida infections continue to be associated with significant morbidity and mortality1. Because of the significance of Candida species in human health, there is a need for readily available molecular tools that allow the study and elucidation of their pathogenesis mechanisms.
One important tool that allows researchers to visualize and quantify microbial cells and the proteins that they express is FP fusion technology. Polymerase chain reaction (PCR)-mediated gene modification, as described in this paper, allows the construction of fusions, between FP sequences and a Candida protein coding sequence of interest at its genomic locus. Stable integration of the construct facilitates analysis of protein expression as well as protein localization dynamics. Plasmids containing FP sequences, optimized for expression in Candida albicans and that can be used in the PCR-mediated gene modification strategy, have been previously constructed2,3,4,5. Plasmids contain FP transformation &#34;cassettes&#34;: a FP sequence linked to a nutritional marker gene that facilitates the transformation of C. albicans and C. parapsilosis2,3,4,5,6,7. Currently available plasmids contain a variety of selectable nutritional marker genes (URA3, HIS1, ARG4) for transformation of auxotrophic strains as well as a dominant drug resistance marker (NAT1), which facilitates transformation of clinical strains lacking auxotrophies. In addition, plasmids contain options for up to four different FP sequences (green [GFP], yellow [YFP], cyan [CFP], and cherry [mCherry]) and either an ADH1 termination sequence for construction of carboxy-terminus protein fusions, or a promoter sequence for construction of amino-terminus protein fusions. Primers are designed with homology to the plasmid DNA surrounding the FP cassette. In addition, the primers also contain 5&#39;-extension sequences bearing homology to the yeast gene of interest to be tagged, which facilitates integration of the cassette into the genomic locus via homologous recombination (Figure 1). Gene-specific FP cassettes are generated by PCR and then transformed into Candida cells made competent for uptake of DNA by treatment with lithium acetate.
<img alt="Figure 1" src="/files/ftp_upload/55333/55333fig1.jpg" /> 
Figure 1: Diagram of how FP sequence fusions are generated in Candida species. (A) Plasmid DNA includes a FP sequence and a sequence encoding nourseothricin resistance (NAT1). Relative locations of Forward (FWD) and reverse (REV) primers are shown, with black portions of the primers indicating the region of homology to the plasmid sequence and the purple portions denoting the gene-specific homology region or primer extension. (B) FP cassettes are transformed into Candida and integrate within the ENO1 genomic locus via homologous recombination (dotted lines). (C) Resulting FP fusion sequence at the 3&#39;end of ENO1. <a href="http://cloudflare.jove.com/files/ftp_upload/55333/55333fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Herein, we present an example of protein fusion (Eno1-FP) constructions in Candida species. We use tagging plasmids containing the NAT1 transformation marker gene along with sequences encoding GFP, YFP, or mCherry (Figure 2). These plasmids are used along with primers in PCR to generate gene-specific cassettes that facilitate fusion of FPs to the 3&#39;-end of ENO1, resulting in expression of Eno1 fused to FPs at its carboxy-terminus.
<img alt="Figure 2" src="/files/ftp_upload/55333/55333fig2.jpg" /> 
Figure 2: Maps of FP cassette-containing plasmids. Forward (F) and reverse (R) primers used to generate the cassettes from the plasmids are indicated along with the relative location of their homology to the plasmids. Primer sequences are as listed in Table 1. F1 and R1 were also used to generate the pYFP-NAT1 cassette. The plasmid containing the YFP-NAT1 cassette (pMG2263) is identical to pMG2120 with the exception of YFP in place of the GFP sequence. Cassette sizes: GFP-NAT1, 3.7 kbp; mCherry-NAT1, 3.2 kbp; YFP-NAT1, 3.7 kbp. This figure has been modified from Gerami-Nejad, et al.4 <a href="http://cloudflare.jove.com/files/ftp_upload/55333/55333fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100W mercury lamp</td>
			<td>CHIU Technical Corporation</td>
			<td>M-100T</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Carrier DNA</td>
			<td>Ambion</td>
			<td>AM9680</td>
			<td>Sheared Salmon Sperm DNA 10 mg/ml</td>
		</tr>
		<tr>
			<td>CCD Camera</td>
			<td>Photometrics</td>
			<td>CoolSNAP HQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Tube</td>
			<td>Corning</td>
			<td>430828</td>
			<td>50ml</td>
		</tr>
		<tr>
			<td>Culture Tube Rotator</td>
			<td>New Brunswick</td>
			<td>2013923</td>
			<td>TC-8, or Any Culture Tube Rotator</td>
		</tr>
		<tr>
			<td>Deoxynucleotides (dNTP) PCR Grade</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Tubes</td>
			<td>Eppendorf</td>
			<td>022363719, 022363212</td>
			<td>0.5ml, 1.5ml</td>
		</tr>
		<tr>
			<td>Erlenmeyer Flask</td>
			<td>Fisher Scientific</td>
			<td>7250089</td>
			<td>125ml</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic Acid (EDTA)</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer (-80 &#176;C )</td>
			<td>Thermo Electron Corporation</td>
			<td>ULT-1386-9-V</td>
			<td>Revco Ultima II</td>
		</tr>
		<tr>
			<td>GFP, YFP and Texas Red Filter Sets</td>
			<td>Chroma Technology Corporation</td>
			<td>49002, 86004v2, 49008</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass culture tubes</td>
			<td>Fisher Scientific</td>
			<td>1496126</td>
			<td>75mm</td>
		</tr>
		<tr>
			<td>HRP goat anti-mouse antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2005</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP goat anti-rabbit antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2301</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator (30 &#176;C )</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Acetate</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysogeny Broth (LB) Media</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5415 D</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>E600</td>
			<td>Nikon Eclipse E600</td>
		</tr>
		<tr>
			<td>Microscope Image Analysis Software</td>
			<td>Universal Imaging Corporation</td>
			<td>6.3r7</td>
			<td>MetaMorph Software Series 6.3r7</td>
		</tr>
		<tr>
			<td>Mouse anti-GFP antibody</td>
			<td>Roche</td>
			<td>11814460001</td>
			<td></td>
		</tr>
		<tr>
			<td>Nourseothricin</td>
			<td>Fisher Scientific</td>
			<td>50997939</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Thermocycler</td>
			<td>Applied Biosystems</td>
			<td>9700</td>
			<td>GeneAmp PCR System</td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>BioExpress, GeneMate</td>
			<td>T-3035-1</td>
			<td>0.2ml</td>
		</tr>
		<tr>
			<td>Polyethylene Glycol 3350</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-mCherry antibody</td>
			<td>BioVision</td>
			<td>5993-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerator (4&#176;C)</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ1500</td>
			<td></td>
		</tr>
		<tr>
			<td>Table Top Centrifuge</td>
			<td>Labnet</td>
			<td>Z 400</td>
			<td>Hermle Z 400</td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td>Vortex Genie 2</td>
		</tr>
		<tr>
			<td>Yeast Extract Peptone Dextrose (YPD) Media</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of fluorescent protein fusions in candida species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

As an example, we used the protocol described above to construct GFP and mCherry fusions to Eno1 in a C. parapsilosis laboratory strain. Each putative transformant was initially restreaked for growth. In this example, since the resultant fusion protein is highly expressed (enolase) and the FPs are bright, we were able to screen transformants by fluorescence microscopy prior to performing diagnostic PCR (Figure 3)6.
<p class="jove_conte...

Watch this Scientific Journal Video about Generation of Fluorescent Protein Fusions in Candida Species at JoVE.com

Generation of Fluorescent Protein Fusions in Candida Species

PCR-mediated gene modification can be used to generate fluorescent protein fusions in Candida species, which facilitates visualization and quantitation of yeast cells and proteins. Herein, we present a strategy for constructing a fluorescent protein fusion (Eno1-FP) in Candida parapsilosis.

Generation of Fluorescent Protein Fusions in Candida Species

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. ...

The overall goal of this procedure is to generate fluorescent protein fusions in Candida species by using PCR mediated gene modification to tag a protein encoding sequence at its native genomic locus, thus ensuring stable integration and expression of sequences over time. This method can be used to generate fluorescently tagged Candida strains and proteins, which facilitates their identification and quantitation for both in vitro and in vivo analyses. The main advantage of this technique is that it's fast compared to conventional cloning approaches and offers the potential to screen transformants by colony or individual cell-based fluorescence microscopy.Though this method was initially developed to tag proteins in Candida albicans, it can also be applied to other yeast species, such as Candida parapsilosis as described in this protocol. Generally, individuals new to this method struggle to obtain transformants that contain correct cassette integrations into the intended gene locus. This problem can be minimized by optimizing conditions and reagents at each procedure step as highlighted in the protocol.After designing primers according to the text protocol, to prepare 500 microliters of a master mix for PCR, combine 320 microliters sterile water, 50 microliters of PCR buffer, 20 microliters of dNTPs, 40 microliters of 25 millimolar magnesium chloride, 20 microliters of purified plasmid, 10 microliters of each forward and reverse primers and 30 microliters of Taq polymerase. Vortex the master mix, then aliquot 50 microliters into each of 10 0.5 milliliter PCR tubes, then place the tubes into the thermocycler and run the following steps. Pool all the products from the 10 PCR tubes into a 1.5 milliliter tube, then subject five microliters of pooled PCR product to agarose gel electrophoresis to verify amplicon size and obtain an estimate of product concentration based on comparison to a DNA ladder.Precipitate the DNA by adding 50 microliters of three molar sodium acetate, followed by 750 microliters of 95%ethanol to the products. Incubate the samples at minus 20 degrees Celsius for at least 30 minutes. Harvest the PCR products by centrifuging the tube at 16, 000 times g for 10 minutes.Carefully remove and discard the supernatant and dry the pellet overnight, then use 40 microliters of TE, pH 8.0, to re-suspend the dried DNA cassette pellet and store the sample at room temperature until use. On day one, recover the yeast strain to be transformed from a 15%glycerol frozen stock. Incubate a two milliliter liquid YPAD culture at 30 degrees Celsius with agitation overnight.On day two, use 50 milliliters of fresh YPAD to dilute 300 microliters of overnight yeast culture in a 125 milliliter Erlenmeyer flask with a breathable cap. Shake the culture at 30 degrees Celsius for approximately three hours. Transfer the overnight culture into a 50 milliliter conical tube and pellet the cells by spinning the tubes in a balanced tabletop centrifuge at 1, 500 times g for five minutes.Pour off and properly discard the supernatant and use five milliliters of water to re-suspend the cell pellet then re-pellet the cells by centrifugation. Remove and discard the supernatant and use 500 microliters of TE lithium acetate to re-suspend the cells, then transfer the suspension to a 1.5 milliliter tube. Centrifuge the tube at 3, 000 times g for two minutes.With 250 microliters of TE lithium acetate, re-suspend the cells. To prepare a negative control, to a fresh tube, add five microliters of 10 milligrams per milliliter carrier DNA and 150 microliters of prepared Candida cells. Incubate the samples at room temperature for 30 minutes.To each transformation, add 700 microliters of plate mix. Pipette the mix up and down gently to mix and incubate them at room temperature overnight. On day three, incubate the transformation mixes at 42 degrees Celsius for one hour.Centrifuge the transformations at 16, 000 times g for 30 seconds, and remove the supernatant, then use 150 microliters of water to re-suspend each pellet by very gently pipetting up and down so as not to damage the cells. For transformations utilizing auxotrophic marker genes such as URA3, plate each total mixture by pipetting the solutions onto the appropriate selective medium agar lacking uridine, and with sterile glass beads, spread the mixture evenly. For transformations using the nourseothricin resistance marker gene NAT1, plate the transformation mixes first onto non-selective YPAD agar and incubate the plates at 30 degrees Celsius for six to 12 hours to aid in cell recovery before nourseothricin stress is applied.After partial growth recovery, replica plate the Candida cells onto YPAD containing 400 micrograms per milliliter of nourseothricin. If the transformation is successful, colonies should appear within one to three days, with no colonies appearing on negative control plate. For auxotrophic and nourseothricin marker selection, streak putative transformants as single colonies onto fresh selective medium agar plates and incubate the cells at 30 degrees Celsius to propagate yeast cells that can be screened for successful construction of FP fusions.As shown here, the protocol demonstrated in this video was used to construct GFP, YFP and mCherry fusions to Eno1 in Candida parapsilosis. The enolase fusion protein is highly expressed and the FPs are bright. Therefore, transformants were screened by fluorescence microscopy prior to preforming diagnostic PCR.In this figure, enolase localization was observed in yeast cells expressing Eno1 fluorescent proteins. The different FP colors were useful to visually distinguish between different yeast strains within a mixture. Proteins were isolated from cell lysates and subjected to SPS-PAGE and western blotting to detect the GFP, YFP and mCherry tagged enolase fusion proteins.All the proteins were detected and were of the correct size. No signal was observed for the untagged parent yeast strain. Once mastered, this technique can be done in less than six hours a day over three days, excluding final incubation days, if it is performed properly.While attempting this procedure, it's important to remember to invest the time to perform verification checks along the way, to ensure that DNA, yeast cells and reagents are optimized. This will increase the chances of a successful transformation.

generation-of-fluorescent-protein-fusions-in-candida-species

Research

JoVE Journal

Genetics

10.5K visualizações.  University of Minnesota. The overall goal of this procedure is to generate fluorescent protein fusions in Candida species by using PCR mediated gene modification to tag a protein encoding sequence at its native genomic locus, thus ensuring stable integration and expression of sequences over time. This method can be used to generate fluorescently tagged Candida strains and proteins, which facilitates their identification and quantitation for both in vitro and in vivo analyses. The main advantage of this technique is t...