Tell me, Flies, tell me, sweet little Flies

I've had this song stuck in my head all day. Simply replace "lies" for "flies" and the song in its entirety is worryingly apt for my research. Give it a go and then I'll give you some context.

I finally managed to delete a gene using CRISPR! So I've been waiting 11 days to see what the homozygous mutants look like.

13 days later (because the food isn't so great) and...

They look absolutely fine.

Bugger. I'll have to wait a few days to see whether they are fertile and a few more until they possibly drop dead or start behaving oddly. Chances are they don't have anything wrong with them which means I'll have to start poking around a little more.

There are several options - a sensible one is to do some genetic interactions with candidate genes (that have a phenotype when mutated) obtained from an interactome I obtained. The caveat there is that it's an interactome for the human protein and not the fly one. Ideally make a mutant phenotype better/worse when adding my mutation to the mix.

Another option is to look a lot closer. Based on expression data there seems to be an enrichment in hemocytes (sort of fly macrophages) and the Central Nervous System. Given I share lab space with experts on hemocyte function and behaviour it seems like it's worth a quick look in that direction. The central nervous system I can delve into with simple things like "is there a difference in size/appearance" but I'd also probably hope to see things go wrong with the flies as they get older.

Anyway, there's a bit of work to be done yet until I find a solid lead. Now I have the mutant I may as well at least establish that it conclusively does nothing before moving on.

So yeah - no, no, no you can't disguise. Tell me Flies.

Science Music

I may have used this one before (Every post is exactly the same) but this is another song that comes to mind in a repetitive screen.

Screened 130 of the 450 potential mutant fly lines now. Out of the 6 different deletions I've got one.  As cruel fate would have it, it's in the gene that lies within the intron of my gene of interest. So I potentially have a control mutant for my non-existent mutant of interest. I guess I can't get too pessimistic until I hit the half way mark. As it is the long weekend out of the country will provide a much needed break as there's no way I can nip into work to do "a few DNA prep/PCRS" while away.

Science Music

This song definitely sums up the last couple of weeks at work.

I guess it's the downside of screening. I'm essentially going in the morning and collecting virgin* flies from 350 crosses, crossing these flies to other stocks, having lunch and then repeating the process in the afternoon. Over and Over again.

I guess it serves me right for picking a gene on the X chromosome. This means I have to go for the "maiden" female flies as there's the possibility the mutation may be lethal which isn't so great if you're a male and only have the one X chromosome to play with . It also means that all the males from the crosses are potentially ones where the deletion hasn't occurred so there's no point in using them at all. I was planning on doing some courtesy PCR on the males in the sense that if there is a deletion I'd know it isn't lethal. I haven't really had the time though.  I'll probably wish I made the time if it turns out it is a viable mutation and I could have just picked a bunch of males from each line ONCE rather than twice a day for a week. Let's not think of that though.

Next week, I can at least change the repetition of virgin collecting (not as fun and/or sinister as it sounds) with serial fly squashing (essentially as sinister as it sounds) and PCRs.
I have another mutagenesis screen on the go too but fortunately that one isn't on the X chromosome so it will be an absolute breeze by comparison. Unless it's not.

*Just in case you think I'm some kind of kinky drosophilist I should point out that flies use internal fertilisation to reproduce (like most people do) and not externally by the female dumping a load of eggs and then the male does his manly duties over said laid eggs. For this reason, when doing crosses you need females that haven't mated with their brothers (flies are cool with Game of Thrones plotlines) or you will wind up with progeny you don't really want.
They do it doggy style in answer to your inevitable follow-up question. In terms of "how do you know they are virgins" - you can do it based on time or you can check if they have a black spot on their belly. If they have a spot they are still virgin. It's definitely a talking point at interviews if you put that one on your CV.

Counting your flies before they've eclosed

A bad case of underestimating the amount of work to set up a CRISPR screen in flies.

The last time I tried CRISPR mutagenesis I got 17 adult flies from one injection and 23 from another. 40 single crosses in total and only 50% were fertile. Which meant the 4 batches of PCR screening per founder line wasn't too traumatic (until the point I discovered there weren't any mutants).

This time I decided to increase the number of injections and different mutant designs. I figured 6 injections wouldn't be too much of an extra workload based on previous runs. I mean it would probably result in 6 x 30 crosses so less than 200. Perfectly manageable. Well, I currently have 300 crosses set up and at least another 100 to set up.

If I think that's painful just wait until I reach the stage where I have to collect virgins from 400 separate crosses. Here's hoping some of them are sterile and that there's a mutant in there somewhere!

Even more fun is the fact I have another 2 injections arriving tomorrow.

A little bit of staggering or crosses at different temperatures may be required.

Time to Try a Technique!

Happy New Year!

I'll try and get back into the swing of things but things have been pretty hectic in terms of priorities (inside and outside work) and while I was wanting to do an update on some exciting experiments, they've been hitting a few bumps and it's hard to not sound negative about negative results! But I really am finding the current work interesting and, dare I say, fun. For those who are wondering - it's CRISPR in flies. It's looking like it's becoming the "in" technique at the moment, largely because it has the ability to do several things such as gene editing, deleting and tagging with (apparently) high levels of efficiency.
I'm really keen on using it for endogenous tagging but I'm still in the process of evaluating how well it works in deleting genes. So far it's 1 gene out of 4 attempts - hence the negative results. I have another batch coming through next week in a make or break attempt (multiple guide RNA combos). Although I'm strongly considering going down the endogenous tagging route on the off-chance it is more successful.

Anyway, trying something new can actually be a great way to stimulate interest in a project and I'd urge others to leap into these things when given the opportunity to do so. My only caveat is that you have to be convinced of all the cool downstream things you can do with it once you have it working. It may not be CRISPR for you but try and find that up-and-coming (or cool-but-somewhat-scary) technique that catches your attention. I know people who have had fun (and a lot of success) with SILAC and a colleague is currently sinking his teeth into APEX with what's looking very promising (and multi-functional in terms of imaging/labelling). I even heard about a technique today that involves using lasers to "detonate" proteins of interest (Chromophore assisted light inactivation/CALI).

My goal is to hopefully have CRISPR set up in the flies so that I (and others from the lab) can pick a novel gene of interest and;

1) delete it/generate a null
2) tag it endogenously with GFP/HA/Cherry (quicker than generating antibodies in theory)
3) replace gene with point mutations (also under endogenous expression)

Those three things should allow me to do a ton of different experiments - assuming step 1 gives something of interest.

It may not be working perfectly but my new year's resolution (besides negating the xmas spread - also not working perfectly) is to to get it working or find valid reasons to ditch it.

You should pick something too; if you're really up for it, develop a technique from scratch rather than use an existing technique. Imagine how cool it would be to develop the next RNAi or CRISPR? And if you do try to avoid winding up as a van driver - unless that's what you want to do!

Office science

This offensive item is currently living in an office that is acting as a temporary kitchen. It seems to be at the stage where no one will risk touching it for fear of an explosion.
The question is how long will the plastic stand up to the pressure? I guess only time will smell ...

Forget Me Not

At around 9:30 pm last night I was about to make some food, open a beer and watch a film, when I suddenly thought "did I take those mini-preps out of the centrifuge after the final spin?". Surely I had, but the doubt lingered and I knew if I didn't nip it in the bud it would ruin the evening and probably culminate in a night where nightmares about DNA visibly decaying in front of my eyes would ruin my sleep. So I went into work. Luckily, it only takes 10 minutes if I jog.
Unsurprisingly, said mini-preps were comfortably nestled inside the freezer. The centrifuge lid was open in what appeared to be a mocking laugh. I walked back home and got a take-away so the trip wasn't a complete waste and nightmares about work were avoided.

Doubt is a dangerous thing.

Mutant Fail

This song pretty much sums up the discovery I made on Friday.

Basically the Drosophila mutant I have (and have been performing various experiments with) is still being expressed, Which isn't great news when you've been assuming it is a null mutant (a mutation with no expression of functional protein).
The mutant is missing the first two exons and has lost it's original start (ATG) site but annoyingly it has chosen to pick up an alternate start site. This gets more tricky as there are two that are close together

ATGCCATGGGC...

If it has picked the first one then I should still be ok as it is out of frame has 8 nonsense amino acids and then hits a STOP codon. The problem is that the second one is in frame and would happily make the remainder of the protein that contains at least one domain that is vital to its function.
The unfortunate thing is that I have no way of telling which start site is being used without an antibody (which I don't have). If anyone knows of a way to do so - I'm all ears. The best I can come up with is to clone the mutant cDNA into a GFP vector and see what is expressed. Not a great experiment as the only thing it can confirm is whether a truncated protein is being produced,

Ultimately I need to make a genuine null mutant which is what my priority has to be. Somewhat worrying with 11 months left on my contract but I may as well get on with it.

In an attempt to see a positive in this revelation - it may at least explain the lack of a phenotype in the mutant I have been using. Maybe the real deal will have a phenotype that rewards the mutant making!

Egg on my face

In the lab today I asked whether there were any really old culture plates in the incubator as this often happens when one of the lab is away for a while.
One of my colleagues replied "No, but there's a bacterial plate with your name on it that looks over a week old in the other incubator".

Serves me right.

Colour Co-Ordinated

Only realised half-way through the day that the purple in my black and purple striped shirt matches my gloves and tube rack. I don't even like purple that much so it's a weird co-incidence.
That said it shouldn't be noticeable as I "always" have a lab coat on when wearing gloves.


In other news - don't flame coverslips in 70% ethanol and accept that cloning never wants to work when you think it should be a slam dunk done-in-one. At this rate it's not going to be a done-in-a-dozen.

Kiss My Ass(pirator)!

Friday afternoon lab fail. The aspirator jar was being cleaned out and rendering one of the tissue hoods unusable. I thought I'd be proactive, clean out the jar and connect it back up to the aspirator. All seemed well despite some confusion over the double jar system. Got a plate of cells out and went to remove the media only for the damn thing to turn into a mass of bubbles.  Culture ruined!

I guess I could have tried to work out how to get it to suck but when this kind of thing happens at this time on a Friday, the best option is to say "F@%k it" and avoid any further disasters. I did at least take the aspirator apart so others wouldn't be victim to it's anti-aspirating behaviour.

Research Reality Check

The undergrad student who is currently working with got an unpleasant insight into the harsh reality of research today. After slaving away transfecting and staining cells for a week, we had a look at the results on a microscope and within the space of 3 hours destroyed what should have been 9+ hours of glorious picture taking. Perhaps even more importantly was the fact there wasn't any good reason for why they had all failed seeming that some of them had worked previously. I think it may be that the cells were a bit off. So tomorrow we will try with new, improved cells.
Today's results may not seem useful but it will be vital if the student ever considers a life in research.

Two days and counting to get a program installed on my computer.

Bristol University takes its computer safety seriously - some might say (eg me) too seriously. I wanted to align some protein sequences to see how similar they are and to do so there's a free piece of software that can be downloaded and installed. Usually it requires a click on "install" after download and within a minute the program is ready to use. Not so at Bristol University - I have to fill out a request for the IT team to grant me permission to click on "install". Two days later and nothing has been done - I'm not holding my breath that it'll be done by tomorrow either.
So thanks to a stupid system I'm going to lose a week wherein I could have done the piece of work instantly. Nice set-up there.

Fortunately a colleague who left has the program on his computer and as it is next to mine I'm using it. Otherwise this would have been a farce.

EDIT: On the quiet I've heard there's a relatively simple work around that I will test tomorrow. I won't say what it is for fear the IT gnomes find out and see fit to make life more difficult by blocking it.

Padawan Training

Imagine a Jedi wearing a lab coat instead of his cloak with their little padawan by their side (with ponytail).

That's sort of what I'm up to at the moment supervising a student's undergrad project. Whenever my PI mentions supervising students in the office I try and avoid eye-contact. Not entirely sure why but my knee-jerk reaction is it'll take me twice as long to do something I could do on my own. The odd thing is that when I do supervise students I wind up really enjoying it.

How come? Maybe I've been lucky in that the students I've worked with have so far* being competent and interested in their project. Most times I actually get them to do experiments while I do something else meaning I get twice as much done and that initial investment pays off.
I think that their interest is the key factor in why I ultimately enjoy supervising.  When I'm caught up in the day-to-day experiments  I tend to  forget that what I'm doing is still interesting and dare i say occasionally fun. When I have a "captive" audience doing experiments with me for the first time I'm reminded of how things work in experiments. A maxi-prep is usually pretty dull but when you explain what's happening at each stage and what you're ultimately achieving it's not so bad. When you explain how you get a fluorescent tagged protein into a cell-line I can't help but think  "this is a clever technique" (obviously not my own invention).

So it does require a bit more effort but it is a nice change of pace to have a student around. Let's hope I haven't jinxed things and that the current one doesn't go all Anakin on me - contaminating everything he touches and laying waste to my little empire.


*need to keep the current one honest!

Lab Grab - Hunger Games

A member of the lab is leaving today. He has a cornucopia of kit and reagents we all desperately crave. Several people in the lab have already asked him "when can we start taking your stuff". We all know the time is fast approaching and we will have what is rightfully ours - at any cost...

I'd say his bench is far more dangerous than the arena - god knows what chemicals he has left on the surface over the last few years.

Keep out

My boss had a great sign on his office door today.

"Writing a grant.

Do not disturb unless you have data on endosomal sorting"

I left him to his own devices.

Lab fail

Since Christmas I've been cursing the fact that I've been sent a fly with a deleted gene only to discover a diagnostic PCR for said deletion reveals the gene is still there. I thought the fly may be on some "subtle" (the dreaded ultrabithorax*) balancer and that I was checking a heterozygous fly. I checked with the lab that sent the fly and they believed it was homozygous. Cue a lot of grumbling about people sending dodgy stocks.

The problem was that the deletion mutant would be really useful so I thought I'd design another set of primers because it just may be the case that I had got an off-target band that was roughly the same size as the band I was expecting. Desperate, I know.

The thing is when I got around to designing the primer I made sure I had the full info on the deletion as well. Eyeballing it, I thought "the deletion looks a lot bigger than the whole gene". That's when I realised I was comparing the genomic DNA of the deletion with the cDNA (the DNA that only encodes the protein). That's when it dawned on me that the primers I had designed for the diagnostic test were cDNA specific. The 1.5kb band I was expecting would be around 33kb from the genomic DNA. Never going to work with the PCR protocol I have!

So thanks to my ineptitude the deletion may well still be there. I've ordered the appropriate primers and will know by the end of the week whether it's real or not. Fingers crossed it is - I'd rather be an idiot than have to try and make a mutant from scratch.

*Ultrabithorax may sound like a really cool Decepticon but in reality is a really hard to distinguish phenotypical marker in Drosophila that only those gifted with "the sight" can easily recognise. The phenotype is present on the haltere of the fly which is like a tiny secondary wing that is used for manoeuvrability.

A) Wild Type  B-D) ubx130/Wild Type.  Figure taken from http://mbe.oxfordjournals.org/content/21/2/348/F3.expansion

You need to keep in mind that the haltere is very small to begin with.

Picture taken from http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/H/HomeoboxGenes.html

Basically I hate it. I'd love it a lot more if the marker was as penetrant as the homozygous mutant I'd love it a whole lot more because the phenotype is super cool.

Picture taken from http://www.bio.davidson.edu/molecular/ubx/ubx.html

Da Vinci Dragonfly!  

EDIT: Fans of "Jonathan Strange and Mr Norrell" should appreciate the footnote to text body ratio in this post :P

Sweet Dreams Aren't Made of These

Had to get out of bed excessively early. Why? Because I was having a reoccurring dream where I was performing RNA extractions and realising I'd done something stupid mid-way. Things like realising I didn't have a glove on or I hadn't washed surfaces with SDS first. A particular favourite was discovering I'd put the flies and Trizol in a 10 ml falcon rendering it impossible to squash them with a pestle and then wondering if i could get away with decanting them into a 1.5 ml eppendorf.

It seems pretty sad when even my anxiety nightmares are science based.

As you may have guessed - I'm planning on doing RNA extractions later in the week. Hopefully these mental trial simulations will mean the real thing goes more smoothly!

Freezer Flood

Returning to work after a week and a half vacation is always tricky. First of all pretty much everything of use from my bench was stolen. Marker pens, tips, scissors, you name it. So I just went and took some more from another colleague who is away. The cycle is never-ending. A rational response would be to just make sure everyone has one of all the essentials but it seems to be a sport in my lab. On Friday I discovered that my bay area was flooded. Even weirder was the fact it was coming from the freezer that doesn't work so I've no idea how it managed to condense enough water to cause a flood. Almost as if it briefly came back to life just to annoy me. Not anymore. I've unplugged it. On the other hand the liquid nitrogen containers were dangerously low. I went to fill it up only to discover that both reserve tanks were also empty (see previous mantra). I took them down to be refilled. I'll check tomorrow to see whether they still need filling. I suspect they will.

The rest of the week seemed to involve repeatedly encountering the last of everything. This really winds me up but I guess the fact I don't personally take over ordering means I'm also to blame. I do try and adhere to the "you take the last one you order more" policy.

The "green" pipette tips have transmuted into gold as we don't have any more. This one is a bit odd as several people have claimed to order them and they've never arrived so I think that one is a distributor problem. The result is that a bartering system has now started in order to get our hands on tips within the lab. I'm also being super efficient with the use of the green tips. If I can use a "red" tip (10ul), I use that and gone are the days when I'd recklessly use a green tip for making mini-preps. I probably should count how many I have left every evening to ensure none of them are stolen. Hell, I should probably take them home with me every night.

Science Music

For those of you who remember my post about performing climbing assays with flies (if not then have a look here) a song has recently come to mind as a potential anthem. It probably required a few months of repeating the experiment for the realisation to occur that Radiohead fits quite well.