Hi all,
I am trying to bombard unc-119 worms with my plasmid construct. I am very new to bombardment, have never done it before. I have few concerns regarding
growing worms and maintaining them.
1)I started with HT1593 worms, grew them on large (10cm plate) enriched peptone plates with streptomycin and fungizone (this was to avoid contamination).
The problem I am facing is that my worms are dying after 4-5 days on the recovery plates. So I have big trouble getting rescued worms as 95% of them die. I
cannot find the reason for this.
2)I was feeding them HB101 bacteria. I observed that on the 2-3 days of putting the bombarded worms on the recovery plates a thick and hard layer forms all
over the plates and soon the worms start to die.
3)the plates also smell really bad. Thinking that HB101 is the problem I started feeding them OP50, but this time also they died.
Till now I have bombarded worms 3 times and all 3 times they died. I could get very few rescued worms, but none of them gave me the transgenic line.
I am really having a hard time maintaining them after bombarding.
Also recently my unc-119 worms (not bombarded) are also showing the same problem. I can not propagate them from 1 plate to 6 plates because they too are
dying.
Has anyone experienced this kind of difficulty while bombarding? any suggestion will be really helpful for me.
Thanks.
I observed that on the 2-3 days of putting the bombarded worms on the recovery plates a thick and hard layer forms all over the plates and soon the worms start to die.
This is not something that happens with either OP50 or HB101. I recommend that you streak out HB101 from frozen stocks, use a single colony to prepare broth and spot some plates, clean your unc-119 strain (by bleaching gravid animals for clean eggs outside the lawn on one of the plates with clean HB101), and repeat the process making sure to eliminate possible sources of contamination.
One possibility is that it’s the streptomycin or fungizone; I’ve not used either, so if they had the effect you describe I’d not know of it. Neither should be necessary if you can maintain a clean work area.
Re: 1) Does this mean you have an EXISTING contamination problem in the lab and if so, have you identified where it comes from; e.g. from the plates themselves (check by incubating a bacteria-free plate) or the bacteria themselves (incubating a streak plate) or from the technique itself (control plates) or from the HT1593 strain you have (clean it up as Hillel suggests) or from your lab environment (do the necessary tests)?
If your plates are sterile and your strain is clean and your technique ok then you shouldn't need antibiotics and antifungal reagents.
You mention a 95% mortality (worms are dying after 4-5 days in recovery), this might be also to do with your biolistic transformation protocol; settings for the gun, post-transformation handling etc. What protocol are you using?
Are you using high purity helium gas?
Re: 2) What did the plates smell like, yeasty or mouldy or like a bad cold? What colour was the film? Did it start from different places on the plate? Is the film flat or sprouting, does it show intesecting boundaries, wow the list is endless-etc. Did you start the HB101 culture from a single colony off a streak plate? Have you tried starting a new culture using an existing frozen stock of HB101 and does this look different?
Re: 3) I assume that aside from the smelly HB101 plates you’re using OP50-1 (the streptomycin resistant variant of OP50) here?
What makes you say that you could rescue some worms but that these were not transformed? Were these worms picked from starved plates and identified through a wt phenotype? In addition to the unc-119 rescue , was the plasmid also carrying a gfp marker?
I have been using Seydoux protocol but with alot of modifications. First of all we use streptomycin in our plate. We wash with PBS +Strep +fungizone. these are the two major
differences. and yes I use the purified Helium tank with PSD/1000 particle delivery system for the bombardment.
in order to find out the cause of the dying. I have done the following steps;
streak out new HB101 and put on enriched peptone plate with strep. Grow unc-119 worms on them.
2)I am also growing unc-119 worms on regular NGM plate with OP50
3)grow unc-119 on regular NGM plate with HB101
and finally grow N2 worms on enriched peptone plate with strep and feed them with HB101.
I am planning to bleach every time the unc-119 worms if I have to propagate them to more plates.
I hope the dying issue does not sustain after this.
Regarding getting the rescued worms. Yes these worms were picked from the plates that had dying worms. The plates were little bit starved. Also the plasmid we decided has a 5
terminal gfp tagged to the gene. I have always gotten rescued worms with wild type movement but none of them had any gfp. I screened them both with PCR designed to check
gfp and also under the fluorescent scope. None of these screening methods confirmed the presence of gfp protein. I do not how they are getting rescued back to wildtype
movement.
It will be good to know how this is happening.
Thank you.