Description

E. coli strain HT115(DE3) has a modified lac promotercontrolling the transcription of T7 RNA polymerase. It is an RNaseIII-deficient E. coli strain with IPTG-inducible T7 polymerase activity

This strain grows on LB or 2xYT plates (and is resistant totetracycline), and competent cells can be made using standard techniques.

Note ontetracycline:

TheTn10 transposon interrupting the rnc14 gene carries a tetracycline resistancegene. Therefore, bacteria should be subjected to tetracycline selection (12.5μg/ml) to maintain the RNase deficiency. However, the transposon is quitestable, as we have not lost it in the absence of selection. Using our protocol(see below and Kamathet al.Genome Biology,2, 1-10) inclusion oftetracycline during feeding significantly decreased the RNAi effect for severalgenes tested, so we recommend that it not be used in culture or in NGM plates duringfeeding using the method below. However, using the method of Timmons,etal.(Gene,263, 103-112), animprovement in feeding results by including tetracycline was reported.

NGMMedia:

Forfeeding plates, use standard NGM agar plus the following ingredients:

Carbenicillinto 25 μg/ml final concentration
IPTG to 1 mM final concentration

Platesare poured fresh 1-3 days before use.

FeedingProtocol(fromKamathet al. (2000) GenomeBiology, 2, 1-10):

1.Pick and grow bacteria 6 hours - overnight (but no longer than 18 hours) in LB+ 50 μg/ml ampicillin, seed onto NGM agar plates including additives (above).(Do not add IPTG or tetracycline to the liquid culture, as this will reduce theRNAi effect.). Bacterial cultures grown shorter times (6 hours) sometimes givebetter results. The lawn quality is improved if the culture is dried quickly byleaving the lids off for about 20 minutes after seeding, but we don't know ifthis affects the RNAi effect. We also have anecdotal evidence that platespoured at least 1 week before use work better than freshly poured plates.

2. Let dry and induce overnight at room temperature.

3. The following day, transfer an L4-stage hermaphrodite onto first plate,minimizing the amount of OP50 bacteria transferred (we usually wash worms in M9buffer and then aliquot them directly onto plates). Leave 72 hours at 15°C (or36-40 hours at 22°C) for RNAi to take effect, then replica plate adult ontoanother plate seeded with the same bacteria. After 24 hours, remove the adultfrom the replica and score the progeny for phenotypes. Alternatively, aliquotembryos or larvae onto the feeding plates and score them later.

4. Note on temperature:
We have observed that some genes give different phenotypes at 15°C vs 22°C;thus, it may be worthwhile to test a given gene using both conditions.

HT115(DE3) genotype
F-, mcrA, mcrB, IN(rrnD-rrnE)1, lambda -, rnc14::Tn10(DE3lysogen: lavUV5 promoter -T7 polymerase) (IPTG-inducible T7 polymerase) (RNaseIII minus).

pQCXIN,pMSCVpuro,hyg,neo,pBABE-puro,pGH112,pWHM3,pPD49_83,L4440,HT115 DE3,pRevTRE-SV40LargeT,FUW-OSKM,pPYCAGIP,

p53-Luc,p27-Luc,pAP1-Luc,pFC-MEKK,pFA2-Elk1,pCDNA5/TO,pBI Tet,pTRE-Tight-Luc,pRevTet-off,on,pGAD424,pGBT9,pACT2 AD,pSos

YCplac22,33,YEplac181,YEplac195,YEplac112,YIplac211,pYRP7,pDR195,pBI101,pCAMBIA1381Z,pCAMBIA1391Z,pSingle-tTS-shRNA,