蛍光タンパク質融合物中の生成

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.