03 November 2007



I've just accepted a position as research assistant with the Broad Institute of Harvard/MIT. I'm going to be a professional scientist!

The lab in which I'll be working is dedicated to creating deletion libraries for a few different bacteria, including Mycobacterium (the pathogen responsible for tuberculosis) and Pseudomonas (an opportunistic pathogen--meaning it tends only to cause problems if you're already sick--that's especially fond of causing respiratory infections in people with cystic fibrosis). Creating a deletion library means deleting a single gene from the bacterial chromosome, and repeating the process for every gene in the genome. So in the end you have thousands of bacterial colonies, each with a different single gene deleted.

What's really great about this particular project is that we'll be making our deletion library freely available to other researchers. So if a researcher somewhere is interested in a specific gene and wants to know what happens when it's deleted, they don't need to go through creating the deletion themselves, they can just look up the gene in our library and request a sample.

How excited am I to be making that kind of contribution to the scientific community?

Pretty damn excited.

But how, you may be wondering, does one delete a gene from a chromosome? The trick is homologous recombination. If two strands of DNA have a region that matches the other in sequence, that region is said to be homologous between the two. In a cell, two homologous DNA regions can line up, break at the same place, switch pieces, and fuse back together; this is called homologous recombination, or "crossing-over."

Let's say the section of chromosome in which we're interested looks like this:

. . . ====[ Upstream DNA ][ GENE ][ Downstream DNA ]==== . . .

We can build a plasmid (little circle of DNA, kind of like a mini-chromosome for bacteria) that has a section looking like this:

. . . ====[ Upstream DNA ][ Downstream DNA ]==== . . .

(For ease of visualization later, DNA from the chromosome is red and DNA from the plasmid is green.) We can stick that plasmid in the bacteria, and hope for homologous recombination between the chromosome and the plasmid. Recombination only occurs in a few of the cells, though, so we need a means of selecting for those cells that do cross-over.

We need two different recombination events to occur, one on either side of the gene. So we'll stick two marker genes on our plasmid: a strength (like resistance to ampicillin, an antibiotic) and a weakness (like a gene that makes sucrose toxic). First we grow the bacteria on plates containing ampicillin; only those cells that have the plasmid (meaning they've had one cross-over event) will survive. Then we take those survivors and grow them on plates containing sucrose; now only those cells that got rid of the plasmid (meaning they had a second cross-over event) will survive.

So after two rounds of selection, the bacterial chromosomes hopefully look something like this:

. . . ====[ Upstream DNA ][ Downstream DNA ]==== . . .

However, that assumes that one recombination event occurred in the upstream region and one occurred in the downstream region. If both events occurred in the same region, then the chromosome could look like:

. . . ====[ Upstream DNA ][ GENE ][ Downstream DNA ]==== . . .


. . . ====[ Upstream DNA ][ GENE ][ Downstream DNA ]==== . . .

So after all that selection, about 50% of our bacteria colonies still have the gene! We therefore need to add one more step: we pick a bunch of bacteria colonies and sequence that part of the chromosome to see if the gene was deleted successfully. The colonies that pass sequencing get added to our deletion library!

So that's how you delete a single gene from a bacterial chromosome. Part of my job will be figuring out how to optimize that process to handle tens or hundreds of genes at once. That means I get to use robots!

This is going to be awesome.

(Oh, and in case you were curious, the opening to this post comes from this Homestar Runner cartoon.)

1 comment:

"the Dude" said...

cute pic "popular neurosurgery"