Mini libraries - Systematic approaches to study peroxisomes made easy

Abstract

High throughput methodologies have been extensively used in the budding yeast, Saccharomyces cerevisiae, to uncover fundamental principles of cell biology. Over the years, several collections of yeast strains (libraries) were built to enable systematic exploration of cellular functions. However, utilizing these libraries experimentally is often labour intensive and restricted to laboratories that hold high throughput technologies. By focusing on a subset of yeast proteins that have a role in peroxisome biology, we created smaller libraries that are more accessible and convenient to use by all labs. Utilizing the available full genome libraries we have established three “mini” libraries that represent all known and predicted peroxisomal proteins and can be rapidly and easily used for complicated screens. Since one of the libraries is built such that it can be easily modified in the tag, promoter and selection, we also discuss how these collections form the basis for creating a diversity of new peroxisomal libraries for future studies. Using manual tools, available in any lab, coupled with few simple genetic approaches, we will show how these libraries can be “mixed and matched” to create tailor made libraries for screening. These yeast collections may now be exploited to study uncharted territories in the biology of peroxisomes by anyone, anywhere.

Introduction

In recent years high content screens have joined the toolkit of cell biology research, enabling functional screening for a diversity of visual traits not easily studied with previous approaches. In the budding yeast, Saccharomyces cerevisiae, (hereafter called yeast) systematic screening approaches have been facilitated by the engineering of several yeast collections (libraries), each containing hundreds of strains in which each gene was either mutated or tagged in a specific manner. Such libraries include the deletion library [1], where all non-essential yeast genes have been removed, the Green Fluorescent Protein (GFP) library [2] where each yeast protein was tagged at its C’ with a GFP moiety, or the SWAp-Tag (SWAT) GFP library [3] where GFP tagging was performed at the N’ of the protein

While such libraries are powerful for screening even in their original form, their utility can be multiplied dramatically by using simple approaches to integrate any genetic trait of choice into the library in a systematic way [4] creating “tailor made” libraries [5]. Such library creation techniques open the door for a diversity of high content screens enabling systematic exploration of cellular traits. However, many of the systematic technologies are laborious or else require robotic, high throughput technologies to access, making them only feasible for labs that have such a setup.

We here bring forward methods that enable these libraries to be used to study peroxisome biology by any lab without the need for anything but simple manual tools. Peroxisomes are dynamic and intriguing organelles that are well known for their function in fatty acid metabolism and handling of Reactive Oxygen Species (ROS) in the cell [6]. However, the function of many peroxisomal proteins is still not known. The main purpose of the protocol presented here is to provide accessibility for the peroxisome research community to the usage possibilities of systematic peroxisome- centric libraries where each known or predicted peroxisome (or peroxisome related) protein, is represented. To this end we have arrayed four such mini libraries that will be made freely available to the community: A peroxisome SWAT GFP library (either under a constitutive or the native promoter), a peroxisome SWAT Cherry library and a mini peroxisome deletion library (Table 1). The mini peroxisome libraries are all prepared in a 96 colonies per plate format, making this protocol easy to handle for both manual and automated screens. Moreover, we will explain in details how to ‘mix and match’ between libraries, enabling personal design of libraries to tailor them to unique biological questions of interest. We hope that the approaches put forward here will make systematic exploration of peroxisome function accessible to any lab that is interested in the cell biology of this fascinating organelle.

Table 1. Mini peroxisome libraries assembled to date.

Library name	Description	Mating type and Selection marker
Deletion-peroxi	A library that contains strains deleted for all known peroxisomal proteins and proteins affecting peroxisome biology. The strains were manually picked from the knockout library [1] of the entire yeast genome and organized on an agar plate in a 96 well format.	MATa G418 resistance
SWAT GFP-peroxi	The basic SWAT library [3] containing all peroxisomal proteins tagged at their N’ with GFP and driven by the NOP1 promoter. The SWAT module can be utilized for tag, promoter or selection swapping.	MATa Uracil autotrophy
SWAT mCherry-peroxi	Built on the above basic SWAT library but swapped to express all the proteins with N’ mCherry fusion driven by the TEF2 promoter.	MATα NAT resistance
SWAT GFP-seamless peroxi	The basic SWAT GFP- peroxi library in which all the native regulatory elements were restored. This library also contains a cytosolic mCherry marker.	MATα No positive selection for the GFP tag. Cytosolic mCherry selection is NAT resistance

1. Materials

1.1. Currently available mini peroxisomal libraries

To facilitate the creation of tailored libraries we have assembled four peroxisome- centric mini libraries, the SWAT GFP-peroxi, Deletion peroxi, SWAT mCherry-peroxi and the SWAT GFP-_seamless peroxi (See details on each library in Table 1). The library that will be used depends on the biological question asked. All libraries are freely available upon request.

1.2. Library maintenance

1.2.1. Growth Media

The libraries are grown on a standard solid media plates for yeast (YPD or SD) supplemented with selections as required.

Note: when adding G418 selection to the SD media 0.1% Monosodium Glutamate (MSG) should be added in order to maintain the correct pH of the media.

1.2.2. Replicating

1. Manual pinning tool (V&P Scientific, VP409) or a robotic replication tool such as the Singer RoTor bench top robot (Singer Instruments) that enables usage of disposable plastic pads for replication

2. Suitable rectangular plates for manual replication (such as the Nunc Omni tray 140156). If using the Singer RoTor, special plates should be acquired (Singer Instruments, PLU-001).

1.2.3. Freezing

1. Polypropylene 96-well freezing plates (Grenier Bio-one).

2. Freezing media made of 15% glycerol in YPD. Note that if the library contains an expression plasmid than it is better to freeze in media that contains the selection.

3. Pierceable Aluminum-foil sealing film (Excel Scientific, AFS-25)

   _inc.) for sealing plates prior to freezing.

1.3. Designing a tailored library based on the SWAT methodology

The SWAT GFP-peroxi library is an acceptor library in which each peroxisomal protein is tagged at its N’ with GFP and expressed under the constitutively active NOP1 promoter. However, the module that is located upstream to the gene can be swapped with a different tag, selection or promoter as needed [3]. The swap is done by a Synthetic Genetic Array (SGA) procedure [4, 7], during which the SWAT GFP-peroxi library is crossed with a donor strain that contains the cassette to be integrated (See http://www.weizmann.ac.il/molgen/Maya/images/SWATmanual(1)(3).pdf for more details). The basic materials needed to perform the swap are detailed in Table 2.

Table 2. Materials required for swapping modules of the SWAT library.

All are freely available upon request.

Material	Description
Donor module plasmid [3]	The template plasmid into which the cloning of the desirable features will be done
SWAT donor strain	The donor strain that has all the traits suitable for SGA (For SGA procedure see section 2.3.2.1) to which the donor plasmid will be transformed
SWAT Peroxi acceptor library [3]	SWAT GFP-peroxi (See Table 1)

1.4. Preparation of libraries for genetic screens

1.4.1. Transferring libraries into liquid media

1. Manual pinning tool (V&P Scientific, VP409) or a robotic replication tool such as the Singer RoTor bench top robot (Singer Instruments).

2. Any 96 well plates that are suitable for cell culture (Nunc).

3. Liquid media of choice with the appropriate selections [8].

1.4.2. Performing a screen

A wide variety of traits can be measured with the mini libraries including fluorescence levels by flow cytometer, biochemical assays by ELISA, growth rate by plate reader or plate assays, and more. Each would require different equipment. We chose to focus on the description of high content microscopic screens:

1. Any inverted fluorescent microscope can be used. Note: when the signal to noise ratio is high, using a spinning disk microscope should be considered.

2. Glass bottom optical imaging microplates (0.17 micrometer bottom) (Brooks Life Science, MGB096-1-2-LG-L).

3. Concanavalin A for immobilising cells for microscopy (Sigma-Aldrich).

1.4.3. Analysis

For analyses any image processing software can be used such as ImageJ that can be downloaded freely to all computer types from this link: http://fiji.sc/.

2. Methods: Handling your library

2.1. Replicate and freeze

The library will be sent to you on a rectangular agar plate that is suitable for either manual handling or for the Singer RoTor. Along with the library you will receive an updated coordinate map (as an Excel sheet) showing the location and identity of each strain on the 96-agar plate. For long-term storage we recommend that the libraries be kept frozen in -80°C in two copies to ensure that no genetic changes occur in the yeast. Upon receiving the library the following steps should therefore be taken in order to secure two frozen copies of it:

1. On the day of arrival: make two copies of each plate onto new agar plates containing the proper selections. This can be done by either a manual tool that is also available in a 96-pin format or by the Singer RoTor.

2. Incubate the new plates over night (ON) at 30°C.

3. For freezing: the cells should be freshly grown before freezing, therefore take the plates from the ON culture. In advance prepare the required amount of 96-well polypropylene freezing plates by dispensing 100ul of freezing media containing 15% glycerol into each well.

4. Replicate the colonies from the agar plate into the polypropylene plate. Note: It is recommended to take as many cells as possible to ensure easy retrieval of strains upon thawing. Note: From the moment that the cells are in 15% glycerol it is recommended to work rapidly towards freezing the cells since the high concentration of glycerol present in the freezing solution may damage them.

5. Cover the plate with aluminium foil and freeze in -80°C.

2.2. Maintain copies

Having secured a frozen stock of the library you can now prepare the copies that will be used for the routine work in the lab. We recommend to keep three copies of the library:

1. The “do not touch copy”.

2. The “replication copy” from which the entire library will be copied to perform screens.

3. The “picking copy” from which single colonies will be picked. To avoid contaminations, either between strains or external, the picking copy should be discarded often (approximately once a month). Therefore a new replica should be made from the “do not touch” copy. Then the stocks will be reorganised as follows:

1. The new copy will become the new “do not touch”.

2. The old “do not touch” will become the new “replication copy”.

3. The old “replication copy” will become the new “picking copy”.

4. The old “picking copy” can be discarded.

   Regardless, some strains show reduced viability on agar in 4°C and therefore we recommend to refresh all copies no more than every three months.

2.2.1. Thaw a copy

It is highly recommended to discard all the copies of the library that are regularly used and at least yearly and thaw a frozen stock to make a fresh replica. This replica should then be used to make all the working copies. Thawing the library is performed as follows:

1. Take the library plate out from the -80°C and incubate for an hour at 30°C. Incubating for an hour ensures thawing of the liquid, this is crucial when using the Singer RoTor, since the pins will go all the way thorough to the bottom of the well to make sure that cells are actually picked. It will not be able to do so if there is ice on the way. However, when picking manually then thawing does not have to be complete before picking.

2. Spin the plates shortly to bring down any condensation on the aluminium cover. This will help avoid cross-contamination. Only then remove the aluminium foil carefully.

3. Mix the well content well and then replicate from the liquid onto an agar plate and grow ON at 30°C.

4. The new agar plate should be used immediately as a source for a new freezing stock to replenish the one that was just used, and also for replicating all the copies that will be kept at 4°C.

2.3. Pick or prepare a library according to the biological question

Before getting started you must decide which of the libraries would be most relevant for your study and how you would like to tailor them. There are a few possibilities to create the sutible library for your needs (Summarised in Figure 1):

Figure 1. An illustration demonstrating the possibilities of using the peroxisomal mini library.

(a) One option is to use an already existing library, such as the SWAT GFP-peroxi in which all the peroxisomal proteins are tagged at their N’ with GFP. In this case, the library can be taken to an automated microscopic screen without further manipulations. (b) The second option is to cross a query strain with an already existing library In this illustration the query strain harbours a donor module plasmid that is made to swap the swappable N’ module of the SWAT GFP-peroxi library. Mating the donor strain with the SWAT GFP- peroxi library gives rise to a new library that carries the whished traits; the figure illustrates the swapping of GFP with mCherry, producing a library in which all the peroxisomal proteins are tagged with mCherry. (c) The third and most complex option is to cross a library with a second library. This type of cross gives rise to multiple plates that carry both the traits of one library as well the traits of the other.

2.3.1. Use an existing library

The simplest way is to use an already existing library. In such a case no additional work is required. For example, if you are interested in studying a change in transcription or expression levels of peroxisomal proteins it would be best to choose a library in which the peroxisomal proteins are expressed under the native promoter such as the SWAT GFP_seamless-peroxi library. On the other hand if you are interested in studying the localisation of the protein of interest, and since some peroxisomal proteins are not highly expressed, then the SWAT GFP-peroxi would be a good choice. If you are specifically interested in the very low abundant proteins, the N’ Cherry-peroxi library would be suitable due to the strong promoter. Furthermore this library is better for screens in which the media to be used contains high concentration of riboflavin that may cause increased levels of autofluorescence when using the GFP channel. Finally, the deletion library can be used to study the role of peroxisomal proteins under specific conditions.

2.3.2. Mix and match - Create your own library

The next level of complexity would be to create your own library that will be suitable for answering a specific question. This will have to be accompanied by performing mating approaches in order to introduce new genetic modifications into the libraries. There are two possibilities of creating a new library:

2.3.2.1. Cross a query strain with a library

The query strain would be a strain carrying all the genetic markers for SGA and the genetic trait that you would like to introduce into the library (See Table 3 for full description of the libraries genotypes and the genotypes of possible query strains). For instance if you are interested in studying the localisation of a peroxisomal protein on the background of the deletion library then you must create a suitable query strain with the tagged protein and cross it with the peroxi deletion library in order to assess the effect of each deletion on the localisation. If you would like to use the swappable SWAT GFP-peroxi library, the query strain is actually a donor strain that harbours the desired module with your tag and selection of choice (see section 2.3.2.2) for further details). Keep in mind the following points when choosing a query strain:

1. Make sure you know the selection needed to maintain your acceptor library.

2. Choose a plasmid that carries a different selection marker for the creation of your query strain. It is important to bear in mind that the MATa-specific promoter (STE2pr) in the SGA compatible strain is driving the Schizosaccharomyces pombe HIS5. Therefore if your query strain will be created by using a plasmid that contains the HIS selectable marker, at the end of the SGA procedure you will be not be able to obtain a MATa library. Similarly, The MATα promoter (STE3pr) is driving LEU2 so if your query strain has a LEU selectable marker then your final library could be selected only to MATα.

Table 3. Genotype description of the peroxisomal libraries and of possible query strains.

Library name and genotype	Possible query strain genotype
SWAT GFP-peroxi his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 hphΔn::URA3::SpNOP1pr-sfGFP-XXX (Full features of the cassette: Linker1-ScCYC1ter-hphΔn-SceI-URA3pr-URA3-URA3ter-SpNOP1pr-sfGFP-Linker2) MAT a	MATα strain that harbours the SGA traits. The most commonly used strain is the one that contains two selection alleles against the diploids as well as two options for selecting haploids: a MATa-specific promoter (STE2pr) driving the Schizosaccharomyces pombe HIS5 (which is the functional homologue of the S. cerevisiae HIS3) and a MATa promoter (STE3pr) driving LEU2. (his3delta1 leu2delta0 lys2+/lys+metl5delta0 ura3delta0 can1Δ::STE2pr-sp HIS5 lyplΔ::STE3pr-LEU2)
SWAT-mCherry-peroxi his3Δlleu2Δ0met15Δ0ura3Δ0 lys+ canlΔ::GALlpr-Scel::STE2pr-SpHIS5 lyp1Δ::STE3pr-LEU2; NAT::TEF2pr-mCherry-XXX MATα	MAT a strain that does not harbour the SGA traits such as strain BY4741
SWAT GFPseamless- peroxi his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 lys+ can1Δ::GAL1pr SceI::STE2pr-SpHIS5 lyp1Δ::STE3pr-LEU2 ; hoΔ::TEF2pr-mCherry::NAT XXXpr-sfGFP-XXX MATα Note: In the seamless library the tagged gene has no selection attached, therefore it is only possible to get to the diploid stage in the SGA procedure.	MATa strain that does not harbour the SGA traits such as strain BY4741

Once you have your query strain ready you can go ahead and perform the SGA (the detailed explanation on how to perform SGA was previously published [5]. The steps of performing an SGA of a library with a query strain are also detailed in Table 4).

Table 4. An outline of the mating procedure of a query strain with a library.

Step	Description	Media	Time	Temp
1.	Prepare a query strain starter in liquid	Liquid media with the specific selection. For example, liquid SD - HIS for query strain that is able to grow on Histidine deficient media	Over night	30°C
2a.	Plate the query strain liquid starter on an agar plate	Agar plates with the specific selections for example SD-HIS	1day	30°C
2b.	Replicate the library of choice to a new agar plate (can be done in parallel to 2a)	Agar plate with the specific selections. For example YPD+G418 plates for the deletion library	1day	30°C
3.	MatingPin yeast from the refreshed query plate and the refreshed lib plate onto a new plate and mix to enable mating	YPD agar plates	1 day	RT
4a.	Diploid selection	Plates that contain specific selection to both the query strain markers and the library markers. For example, SD+G418-HIS is used for diploid selection of mating between the deletion library and a query strain. Note: SD containing G418 must have Monosodium Glutamic acid instead of Ammonium sulfate to retain a basic pH.	1-2 days	30°C
5.	Sporulation	Nitrogen starvation plates. Note: Do not wrap plates; oxygen is essential for this step. Note: Keep the plates humid by wrapping in slightly wet paper towel in a box.	5-7 days	RT
6.	Haploid selection	SD-His-Arg- Lys+canavanine+thialysine plates to select for MATa haploids or SD-Leu-Arg-Lys+canavanine+thialysine plates to select for MATa haploids. For example, the library created by crossing a query strain that is able to grow on SD-HIS and the deletion peroxi library should be selected to MAT α. Note: when picking colonies from Sporulation plates make sure to touch the colonies multiple times to make sure that as many spores will be transferred to the haploid selection plates.	2 days	30°C
7.	Double mutant selection Final step	SD-His/-Leu and -Arg-Lys+canavanine+thialysine+selection for all markers from both the query strain and the library	1 day	30°C

Once the SGA has been completed (three weeks) you should quality control the newly formed library to ensure that it has undergone the SGA successfully. Quality controlling is performed by two ways:

1. Perform visual check: For final libraries that contain a fluorescent tag, pick eight colonies and look at the microscope to check for fluorescence.

2. Perform check PCR: For final libraries that contain a deletion/mutation, pick eight colonies and perform a check PCR to see that they still harbor the trait of choice.

3. Perform linkage analysis: For each gene in the query strain that will be selected for by the selection cassette that is linked to it (your trait of choice, the Δcan, the Δlyp) it is not possible to obtain haploids that contain these genes from both parental strains and therefore resulting haploids should be dead (for example Δcan1/CAN1-GFP strains can not be made). Due to the relatively low levels of meiotic recombination this also holds true for approximately five gene loci on each end of the selected trait. For such combinations you should expect smaller colonies or no colonies at all on your SGA plate.

2.3.2.2. Cross a library with a second library

An extension of the above possibility is to systematically cross two libraries with each other. Theoretically the crossing of two 96 well plates will produce 96 plates but it is also possible to use a 384 format plates to reduce the amount of plates produced. Depending on the chosen libraries it could be that in order to cross two libraries you will need to transform one library to an opposite mating type. This can be done by taking the library through an SGA procedure with a compatible query strain and then selecting for haploids that are of the opposite mating type and also include the selection markers required for SGA. Crossing two libraries is difficult to perform manually but can become essential if you wish to create a genetic interaction map to learn about genetic interactions between genes (such as synthetic lethality). In this case the two libraries to be crossed are the original deletion peroxi (mating type a) with the deletion peroxi in which the mating type was switched to alpha by an SGA procedure.

2.3.2.3. Using the SWAT library to study peroxisomes

The SWAT GFP-peroxi library has several inherent strengths for the study of peroxisome biology. First it enables visualization of nearly all known peroxisomal proteins in their correct localization when cells are grown on glucose. This is because each gene is expressed under a constitutive promoter (hence even proteins that would be expressed only under specific growth conditions such as growth on oleate, are expressed in glucose). Importantly, the N’ GFP tag has been shown to enable correct targeting of proteins containing a C’ Peroxisome Targeting Signal 1 (PTS1) as well as proteins containing an N’ PTS2 signal [3]. Second, the SWAT module offers the possibility of excising the constitutive promoter and replacing it with another promoter. For example, you could express genes that are important for peroxisome biogenesis under the regulation of an inducible Gal promoter, turning them into “shut off” alleles when cells are grown on glucose. Finally, the SWAT tag enables integration of any other tag of choice (for affinity purification, for split fluorophore labelling etc.) in one simple SGA procedure. For replacing the promoter, the selection and/or the tag, there is a simple SWAT procedure. A query strain is first built on the background of a SWAT donor strain (freely obtainable upon request) that harbours all the required genetic markers for SGA approaches as well as an inducible I-SceI restriction enzyme that facilitates high efficiency of swapping. Second, a plasmid must be created that has the specific linkers for the swapped region flanked by I-Sce1 restriction sites with the tag and selection of choice encoded between them. The plasmid to be used can be either obtained from an existing collection of plasmids or cloned (for a list of available plasmids as well as cloning instructions into the SWAP module see: (http://www.weizmann.ac.il/molgen/Maya/images/SWATmanual(1)(3).pdf)). Note: In case of cloning a new cassette into the plasmid, it is important not to use any of the following selections: URA (the SWAT library marker), HIS, LEU, LYS, and ARG, as well as the CAN1 and LYP1 transporters that are used for the SGA approaches.

Note: A query strain would be an SGA suitable strain of MATα (See Table 3). The main steps of the SGA procedure for swapping are as follows (a step by step description is given in Table 5):

1. Mate the SWAP GFP-Peroxi library with a donor strain to create a heterozygous diploid library that contains both the donor strain genotypes as well as the genotype of the SWAT GFP library.

2. Select for diploids

3. Sporulate in order to produce haploids from which either MATa or MATα will be selected.

4. Select for double mutant haploids that contain both traits of the donor and the library.

5. Induce I-SceI activity by replicating the library onto liquid or agar containing 2% galactose. Let cells grow on galactose-supplemented agar plates ON.

6. Select for swapped yeast by adding 5-FOA that is toxic to all those cells that still harbour the original SWAT cassette containing URA3.

   Note: As a control the original donor strain and strains from the library should be plated, and then transferred to the swap selection to verify that they do not grow (as swapping does not occur in the library, and the donor plasmid should be cut in the donor).

Table 5. An outline of mating a SWAT library with a SWAT donor.

Step	Description	Media	Time	Temp
1.	Donor strain liquid starter	Liquid media with proper donor selection	Over night	30°C
2a.	Plating the donor strain	Agar plates with proper donor selection	1day	30°C
2b.	Plating the SWAT GFP-peroxi	SD-Ura	1day	30°C
3.	Mating	YPD	1 day	RT
4a.	Diploid selection	SD-Ura+donor selection	1-2 days	30°C
4b.	Second diploid selection	SD-Ura+donor selection	1-2 days	30°C
5.	Sporulation	Nitrogen starvation plates. Note: Do not wrap plates; oxygen is essential for this step. Note: Keep the plates humid by wrapping in damp paper towel in a box.	5-7 days	RT
6.	Haploid selection	To select for MATa haploids: SD-Ura-His-Arg-Lys + canavanine + thialysine Or To select for MATα haploids: SD-Ura-Leu-Arg-Lys + canavanine + thialysine	2 days	30°C
7.	Double mutant selection	SD-His/-Leu and -Ura-Arg-Lys + canavanine + thialysine + specific donor selection	1 day	30°C
8.	IScel induction	YEP Galactose (2%)	1 day	30°C
10.	Swap selection	SD-His/-Leu and -Arg-Lys + canavanine + thialysine +5-FOA For seamless tagging +NAT for maintaining the cytosolic cherry.	1 day	30°C
11.	Second swap selection	The same as in 10	1 day	30°C

2.4. Screening a library

In order to microscopically screen the libraries they should be transferred to liquid growth media suitable for the required strains. For instance if it is the SWAP GFP-peroxi library that you are screening then the media you should use is SD-URA. The libraries can be transferred to the liquid media using either the manual pinning tool or the RoToR:

1. Replicate a library from the “replication copy” and grow ON.

2. Pick the library into 100μl appropriate media in a 96-well plate and grow to stationary phase at 30°C.

3. Back dilute the cells in the morning by taking 10μl from the ON culture into 190μl of media in a new 96 well plate and grow to log phase.

   Note: If your assay requires growth in a media other than the growth media you can either grow it ON in the required media or back dilute into it. We advise not to change media after the back dilution as this may require centrifugation of the plate, removal of the growth liquid and re-suspension.

4. When cells have reached mid-logarithmic growth (depending on media this can take between 4 hours in YEPD and longer in Oleic acid), take 50μl of the cells and transfer into a glass bottom Micro Well Plate covered with ConA.

   Note: To prepare the ConA-covered plates, dispense 30μl of ConA at a concentration of 0.25 mg/ml in DDW, incubate for 30 min at room temperature (RT), then remove by aspirating and wait until completely dry.

5. Incubate the cells in the ConA covered wells for 20 min. Wash the settled cells once with synthetic clear media and finally cover with 50 μl of clear media.

6. Before visualizing the sample, clean the bottom of the plate with lens cleaning tissue (Olympus) and 100% ethanol. For the screen it is possible to use any fluorescent microscope at x60 magnification. If the microscope has an automated platform and acquisition software that allows it to take images of the wells in a multi-well plate automatically then this can be used to simplify the screening procedure.

Note: For some assays media turbidity (such as that formed in media containing oleate) can interfere with the readout. This is especially true for measurement of OD₆₀₀ or microscopy in the GFP channel.

Note: If you will require quantitative analysis we suggest to take enough images per well to ensure acquisition of ~1000 cells. Depending on the density of your cells, the camera and field of view this may be around 3-4 images.

2.5. Analysis

If you are using a microscope to screen the library, then the output is simply images. Images can be opened as stack in ImageJ (Fiji) and analysed by either manual inspection for qualitative readouts or simple image analysis program for quantitative analysis such as CellProfiler (http://cellprofiler.org/)

2.6. Confirming Hits

A screen of a library has the potential to give rise to false positives mainly due to a contamination or secondary mutation in the strain. Therefore it is important to verify the direct causality of the genotype to phenotype before moving forward with strains that led to the phenotype of interest (hits). There are a few tests you could do to verify your hit:

1. Rescreen: Pick the positive strains from the library and grow them on a separate plate. Rescreen and make sure that the phenotype appears again.

2. Verification A: Perform a check PCR to verify that the hit genotype harbors the genotype that it should (for example that a certain gene is deleted or tagged).

3. Verification B: Create the same strain separately again, for instance if you found that the deletion of PEX5 affects the distribution of certain proteins then take the strain that expresses some of the perturbed tagged proteins from the SWAT GFP-peroxi library and manually delete PEX5 in these backgrounds. Reimage and verify that the phenotype sustains.

4. Prove causality: In some cases a deletion of a gene affects genes in its neighboring chromosomal context or the phenotype may be the result of a secondary suppressor mutation. To prove causality it is essential to perform a rescue experiment by restoring the activity of the hit gene. For example, returning the activity of PEX5 to the strain that was described in the previous paragraph. This can be done in two ways – either by introduction of a plasmid carrying the PEX5 gene or by creating a strain with an inducible or repressible promoter driving your causal gene and showing that the phenotype is dependent on the activation or repression of the locus.

Concluding remarks

The protocols presented here outline the procedure of using mini peroxisome libraries to study peroxisome biology. We have shared our experience and knowledge to provide you the tools to create and screen a library that will be most suitable for your study. We hope that you will find this manual useful and applicative.