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Monday, May 16, 2005

Don't shoot the messenger!

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Oh whoopie do! I have another "progress" meeting with SWMNBN on Wednesday morning. Those of you who read this blog regularly will know that I look forward to these meetings with the same excitement and anticipation that prisoners at Abu Graib would experience upon seeing Private Lyndsey Walker striding across the courtyard carrying a black sack, a doglead and a set of car jumper cables...

This month, it will be even more fun than usual. Having rather selfishly decided to go for a job interview, I have had less working days than the 20 calender days since the last meeting would suggest. I haven't heard back from the interview yet, so I don't know if I will mention this to SWMNBN. I didn't ask her for a reference for obvious reasons, and she may not be too impressed. On top of that everything that could go wrong, has gone wrong. Basic procedures that are all but infallible have not worked and problems that I thought I had solved months ago have suddently reappeared.

So far, so normal then. The real kicker this month though is not a problem of my making - but one that I am almost certain to cop some (or all) of the shit for. You see I have just proven that the postdoc whose work I rather unwillingly "inherited" a few months ago and have been struggling to progress - fucked up. I mean really fucked up. As in the last 2 years may have to be binned and the entire raison d'etre of the project completely gone.

Oh dear.

Basically, in a nutshell, the postdoc in question (who is now no longer even in the UK) was tasked with removing a number of genes from a strain of brewer's yeast. The perennial problem with anything that involves adding or removing DNA from an organism, is how do you know when you have done it correctly? Contrary to popular belief, you can remove a remarkable number of genes from most organisms without any obvious immediate effect. The effects may take time to develop, or may only manifest themselves under obscure and unusual conditions that are difficult or time-consuming to test for, particularly when the success rate for most gene additions/removals is 1 in 100 or 1 in 1000 or less. Therefore, a common method is to use an antibiotic resistance marker - basically, you swap the gene you want to remove for another gene (known as a "marker") that makes the organism resistant to a normally poisonous antibiotic. Only cells that have successfully swapped the target gene for the antibiotic resistance marker can grow in the presence of the antibiotic. Simple, straightforward and used the worldover thousands of times a day.

However, what if you want to remove 2 genes? The cell has already been made resistant to the antibiotic, so how can you tell that a second gene has been replaced with the antibiotic resistance marker? You have 2 options - first, you can use a second antibiotic resistance marker - in bacteria ampicillin is commonly used, but tetracycline or neomycin might also be used so that you can examine 3 genes simultaneously. The second option is to "recycle" the marker - that is remove it from the organism (this won't magically restore the gene that you originally removed), making the organism susceptible to the antibiotic again, then use it again on a second gene. This is the method we prefer.

Well, for the past 2 months I have been trying to remove the marker that the first postdoc inserted to use it again on a second gene. Despite following the instructions left by the previous postdoc, looking up the relevant protocols on the internet and talking at length with someone who has used the system extensively - I can't remove the marker gene. The cells still grow like weeds on the antibiotic. Everyone agrees that I am doing the right thing. So today I did an experiment to check that the previous postdoc had actually inserted the marker gene correctly. Its' successful removal is critically dependent on it having been designed and correctly inserted in the first place and it took over a year to do it. According to the results of my experiment, the original insertion was fatally flawed. Instead of being removed, the marker gene is just switching its orientation - which unfortunately has no effect whatsoever on its effectiveness.

Short of going back to the very beginning (it took the original postdoc 12 months - I have 2 months left on my contract), I have precious few options. The result of this is that SWMNBN is going to do her fucking nut. And with no one else to vent at, who do you think is most likely to get it in the neck?



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