Southern blot analysis.
DNA was prepared using the standard chloroformd/isoamyl alcohol
technique. This DNA was then digested overnight using PstI.
10 µg of the digested DNA of each clone was subjected to
an overnight migration in a TBE gel. The DNA was then transferred
from the gel to a Hybond-N+ membrane (Amersham). The DNA was fixed
on the membrane 2 hours at 80 °C. The probe was the EcoRI
fragment of the mini-Tn5Cm transposon. This probe was radio-labelled
with CTP32 using the Prime-a-Gene kit (Promega). This radio-labelled
probe was hybridised on the membrane using the ExpressHyb solution
and conditions (Clontech).
Proliferation of Db11 within the worm intestine.
NGM plates were inoculated with an overnight culture of Db11
and incubated at 37 °C for 8-10 h. Plates were seeded with
500 L4 stage hermaphrodite strain N2 worms. Worms were fed on
Db11 at 25 °C for 6 h, harvested, washed 5 times in M9 buffer
and deposited at 25 °C on OP50 containing plates. Each day,
20 worms were mechanically disrupted in LB media as previously
described by Aballay et al. (2000) and the colony forming
unit within their intestine was estimated using LB agar plates
containing tetracycline and kanamycin.
Selection criteria.
During the first round of screening, each clone was tested four
times. All the control worms infected with Db11 were generally
dead at day 10. Of 2,300 clones tested, 11 supported the growth
and survival of worms for at least 15 days (in at least one test)
7D1, 7E7, 7F1, 8E2, 8E11, 8G1, 8H1, 10E5, 10F7, 10H1 and 23C11.
A further 119 supported the growth and survival of worms for at least 13 days (in at least one test) or 12 days (in at least two experiments). These clones were subjected to a second round of screening using the same protocol.
We finally selected among the 119 Db11-derived mutant clones those that during the second round of screening
- supported the growth and survival of worms for at least 13 days (in at least one test) or 12 days (in at least two experiments):
3H5, 7A8, 8C7, 18F3, 21C4, 22D4, 22D9, 23E6.
- those with a cummulative 'attenuation index' (AI) > 3:
10H4, 18D4, 20C2, 21C1.
The cummulative AI is the sum of the AI for the four independent trials. The AI is calculated as follows:
AI = Maximal survival (days) - 10
Reference
Aballay, A., Yorgey, P. and Ausubel, F.M. (2000) Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans. Curr Biol, 10, 1539-1542.