Culturing and Maintenance of RNAi-Expressing Bacteria

Abstract

Source: Anderson, Q. L., et al., A High-throughput, High-content, Liquid-based C. elegans Pathosystem. J. Vis. Exp. (2018)

This video demonstrates the preparation of RNA interference (RNAi) bacteria carrying plasmids to inhibit target gene expression. It outlines RNAi bacterial selection, growth, and induction steps to produce dsRNA-expressing plates for downstream assays.

Protocol

1. Preparation of RNA interference (RNAi) Bacteria

1. Streak out or pin-transfer desired strains of RNAi-containing bacteria onto lysogeny broth (LB) agar plates containing appropriate antibiotics (carbenicillin at 100 µg/mL and tetracycline at 15 µg/mL for Ahringer and Vidal libraries) from a frozen stock.
   Note: When working with nonpathogenic bacteria, use either a biosafety level (BSL)-2 A/B biological safety cabinet or a BSL-1 laminar flow hood to ensure sterility of the cultures and to minimize the chance of cross-contamination of the library.
2. Incubate plates for 24 h at 37 °C.
3. Store the plates at 4 °C for up to two weeks.
   1. After two weeks, discard the plates and replace them with freshly made plates.
4. Test multiple RNAi strains in parallel by individually inoculating a single colony from each clone into 4 mL of carbenicillin-supplemented LB in a single well of a 24-well deep-well plate or into a sterile test tube.
   Note: Tetracycline has been reported to reduce the efficiency of RNAi. Do not use it in this medium.
5. Place 24-well deep well plates into a shaking incubator optimized for multiwell plates and incubate at 37 °C for 16 h while shaking at 950 rpm. Note: It is hard to obtain uniform bacterial growth in multiwell plates using a conventional shaking incubator. The shaking incubator needs to be able to shake at a minimum of 750 rpm in order for the cultures to grow properly. In the absence of a specialized shaker, it is possible to grow each culture in an individual tube. Cultures can be transferred into the 24-well plate for centrifugation afterward.
6. Collect bacteria by centrifuging for 5 minutes at 2,000 x g.
7. Decant the supernatant by inverting the plate and shaking vigorously. Resuspend RNAi bacteria in 100 µL of S Basal (5.85 g sodium chloride, NaCl; 1 g dipotassium phosphate, K₂HPO₄; 6 g monopotassium phosphate, KH₂PO₄ dissolved into 1 L of ultrapure water and sterilized by autoclave).
8. Pipette resuspended bacteria into an appropriate number of wells of a multiwell NGM plate (Nematode growth media, 3 g NaCl, 2.5 g peptone, 1 mM calcium chloride, CaCl₂; 1 mM magnesium sulfate, MgSO₄; 25 mL of phosphate buffer (amount per liter: 132 mL of K₂HPO₄ (1 M) and 868 mL of KH₂PO₄ (1M)), and 18 g agar per liter of water) supplemented with IPTG (Isopropyl β-D-1-thiogalactopyranoside; 1 mM final concentration) and carbenicillin (100 μg/mL final concentration).
   1. Use 3.5 mL of NGM media per well of a 6-well plate, 1 mL per well of a 24-well plate, or 150 µL per well of a 96-well plate.
   2. Use 100 μL/well of bacteria for a 6-well plate, 50 μL/well for a 24-well plate, or 20 μL/well for a 96-well plate. Allow to dry.
      Note: Drying is normally performed in a sterile flow hood to promote rapid, uniform drying. Uniform drying is very important. Avoid over-drying, as cracks in the agar promote burrowing of worms. This leads to worm losses and potential clogging of liquid-handling systems.
9. Use the prepared plates immediately or store them at 4 °C for up to two weeks for later use.
   Note: To prevent plates from drying out, they can be sealed with plastic paraffin film or placed in a tightly sealed container with damp paper towels.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24 deep-well RB block	Thermo Fisher Scientific	CS15124
Nematode growth media (NGM)			Amount per liter: 18 grams agar, 3 grams sodium chloride, 2.5 grams Peptone, 1 mL calcium chloride (1 M), 1 mL magnesium sulfate (1 M), 25 mL phospate buffer, and 973 mL of milli-Q water
Lysogeny Broth (LB)	USBiological Life Sciences	L1520
S Basal			Amount per liter: 5.85 grams sodium chloride, 6 grams monopotassium phosphate, 1 gram dipotassium phosphate, and 1 liter of milli-Q water
Agar	USBiological Life Sciences	A0930
Sodium chloride	USBiological Life Sciences	S5000
Peptone	USBiological Life Sciences	P3300
Calcium chloride	USBiological Life Sciences
Magnesium sulfate	Fisher Scientific	M63-500
Phospate buffer			Amount per liter: 132 mL of dipotassium phosphate (1 M) and 868 mL of monopotassium phosphate (1 M)
Monopotassium phosphate	Acros Organics	7778-77-0
Dipotassium phosphate	USBiological Life Sciences	P5100