Handling of Bacteria From the C elegans RNAi Feeding Library

Gene knockdown via dsRNA mediated interference, or RNAi, is extraordinarily efficient in C. elegans. To generate this knockdown effect, worms can simply be fed bacteria designed to produce dsRNA homologous to a single predicted gene in the C. elegans genome (9,10). In these bacteria, an ampicillin-

Fig. 1. Schematic representation of the genotype of double-stranded RNA (dsRNA) producing bacteria. Strain HT115(DE3) contains an isopropyl-P-D-thiogalactopyra-noside (IPTG)-inducible T7 RNA polymerase within an integrated Tn10 transposon that confers tetracycline resistance to bacteria. An ampicillin-resistance plasmid containing genomic DNA homologous to a single C. elegans gene is flanked by two T7 RNA polymerase binding sites. When exposed to IPTG, the bacteria express T7 polymerase, which results in the production of dsRNA homologous to the DNA inserted between the T7 polymerase sites. When worms eat these bacteria, they lyse and release dsRNA. Released dsRNA is absorbed thorough the gut where it acts to silence gene expression. In a poorly understood process, this silencing effect is rapidly spread throughout the organism to silence gene expression in other tissues.

resistant plasmid contains gene-specific DNA flanked by two T7 RNA polymerase promoters. These bacteria also harbor an integrated, tetracycline-select-able transposon construct containing an IPTG-inducible T7 RNA polymerase. Therefore, bacteria only produce dsRNA in the presence of IPTG (Fig. 1).

A renewable bacterial library containing 16,757 bacterial strains, each producing dsRNA homologous to a single predicted gene in the C. elegans genome, is commercially available (www.hgmp.mrc.ac.uk/geneservice/index.shtml). Methods for replicating and utilizing this library are described in Subheadings 3.2.1. and 3.2.2. This includes methods for replicating and storing the library and manipulating the library for a large-scale genome wide RNAi screen.

3.2.1. Replicating and Storing the C. elegans RNAi Feeding Library

The RNAi bacterial feeding library contains approx 16,757 bacterial strains, representing about 87% of the predicted C. elegans genome. This library is supplied in 55- to 384-well plates, each well containing a single bacterial clone producing dsRNA targeting one C. elegans gene (see Note 4). To prevent contamination of the library, at least one copy should be created and used as the working copy. The original copy should be stored in a separate location and only used in emergencies.

1. Distribute 75 ^L of sterile LB media containing 25 ^g/mL carbenicillin and 8% glycerol into each well of 55-384 well plates using a microfluidic media dispensor. Label each new plate to correspond with the master plates (i.e., Chromosome I, plate 1, Chromosome I, plate 2, and others).

2. Thaw each 384-well master plate to room temperature and place it within the pin replicator alignment tool. Place a 384-well plate containing media only into a second pin alignment tool.

3. Sterilize the 384-pin tool by submerging the pins in bleach, washing the pins twice in ddH20, once in 100% EtOH followed by flame sterilization (pipet tip box lids can be used to hold solutions; use extreme caution when working with EtOH near a flame).

4. Allow the pin tool to cool for approx 5 s, and then insert the pins into the master plate using the pin alignment tool. Inoculate the copy plate by inserting the pins into the second clean plate.

5. Seal the master plates with parafilm and place them back at -80°C.

6. Grow the copy plates overnight at 37°C and then seal them with Parafilm. Store the copy plates at -80°C in a separate location from the master plates (in case of catastrophic freezer malfunction).

3.2.2. Expansion of the RNAi Library

From 384-Well Plates to 96-Colony Omnitrays

Performing a genome wide RNAi screen requires expansion of the frozen bacterial RNAi library from a 384 format to a 96-colony Omnitray format to facilitate physical manipulation of the bacteria (see Note 5). In some cases, bacteria may also need to be streaked to single colonies. For example, we found that for studies of the osmotic stress response, the strongest RNAi-induced phenotypes were observed when cultures were initiated from single colonies as compared with cultures initiated from large bacterial inoculums or directly from frozen glyc-erol stocks. The bacteria are cultured in 96-well plates before being spotted onto 24-well nematode growth media (NGM) RNAi plates (NGM plates [11] containing 25 ^g/mL carbenicillin and 1 mM IPTG). NGM RNAi plates can be poured in very large batches using a sterile media dispensor; 5 L should produce approximately seventy-five 24-well plates, which is enough to screen 1800 genes.

1. Thaw a 384-well copy plate and place it into the replicator pin alignment tool.

2. Place an omnitray containing 40 mL of LB agar containing 25 ^g/mL carbenicillin and 12.5 ^g/mL of tetracycline into a second pin alignment tool.

3. Sterilize the 96-pin tool as described in Subheading 3.2.1., step 3.

4. Place the 96-pin tool into section "A" of the 384-well plate.

5. Allow the 96-pin tool to rest on top of the LB agar containing Omnitray, transferring a small amount of bacteria culture. Do not place pressure on the pin tool, as this will force the pins into the agar.

6. Sterilize the pin tool and repeat steps 4 and 5 for sections B, C, and D of the 384-well plate, resulting in four Omnitrays/384-well.

Fig. 2. Schedule for performing a feeding-based genome-wide RNA interference (RNAi) screen. A strategy for performing two RNAi screens per week is shown. Worm preparation and culture are described in Subheading 3.3.1., and methods for bacterial preparation and culture are described in Subheading 3.2.2.2. During days 2, 3, 6, and 7, other screen tasks should be performed, such as logging data, streaking bacteria, pouring plates, and others.

Fig. 2. Schedule for performing a feeding-based genome-wide RNA interference (RNAi) screen. A strategy for performing two RNAi screens per week is shown. Worm preparation and culture are described in Subheading 3.3.1., and methods for bacterial preparation and culture are described in Subheading 3.2.2.2. During days 2, 3, 6, and 7, other screen tasks should be performed, such as logging data, streaking bacteria, pouring plates, and others.

7. Place the Omnitrays at 37°C overnight and then store the plates containing bacterial inoculums at 4°C.

3.2.3. Culture of RNAi Bacteria for Genome-Wide RNAi Screening

1. Streak each bacterial colony onto one quadrant of a 10-cm LB agar plate (four bacterial strains per plate) containing 25 Mg/mL carbenicillin and 12.5 Mg/mL of tetracycline using standard microbiological procedures (8). Grow the colonies at 37°C overnight.

2. Using an 8-channel pipet, add 100 mL of LB media containing 25 Mg/mL carbenicillin to each well of a 96-well plate. Label these plates to correspond to the Omnitray plate number (i.e., Chromosome I, Plate 1A, 1B, 1C, and others).

3. Pick a single colony from location A1 on the Omnitray into well A1 of the 96-well plate. Repeat for each well.

4. Grow the 96-well cultures for 4-8 h at 37°C (shaking is not necessary).

5. Spot 20-40 m-L of bacterial culture per well onto a 24-well NGM RNAi plate and allow the cultures to dry in a sterile flow hood for 10-20 min.

6. Incubate the plates overnight at room temperature to allow the bacteria to produce dsRNA. Plates containing RNAi bacteria should be stored at 4°C and used within 1-2 wk.

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  • Camilla
    Which bacteria can c. elegans eat?
    7 years ago

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