I’m Frank Schwach. I'm a computational biologist in the Plasmogen team,
which is part of the Malaria programme at the Wellcome Trust Sanger Institute.
I use computer programming to design DNA vectors that can manipulate genes in malaria parasites.
A vector is a DNA molecule that we use to manipulate the genome of the parasite in a specific way.
We can delete a gene. We can modify a gene to do something else. We can change the way it's being used by the parasite.
When we knockout a gene we don't just delete the gene we need to replace it with something.
We bring in a little package of DNA that conveys new functions to the mutant cell. We call this a resistance marker.
The resistance markers are guided into the right place in the malaria genome by short DNA sequences called oligonucleotides.
The design software identifies the best oligonucleotides for each individual gene.
Once the software has designed oligonucleotides it will produce order sheets for them
which will be sent to external companies to actually produce the oligonucleotides.
Once we receive these custom-made oligonucleotides, the lab based stage of the process begins.
This has many steps and takes about 2 weeks.
Firstly the oligonucleotides are mixed with template DNA and a PCR reaction is performed.
Now each well contains DNA fragments with the same resistance marker genes - known as a resistance cassette -
but the ends of these fragments differ and those will later guide the finished vector to its specific target gene in the parasite.
These ends are the oligonucleotides we designed earlier.
Next a crucial step called recombineering happens in bacteria.
Each of these bacterial cultures harbour a specific short piece of malaria DNA
that contains an individual target gene identified at the design stage.
The product of the earlier PCR reaction is then shot into these bacterial cells using an electroporation machine.
Under specific culture conditions the cells now perform the recombineering reaction,
which replaces a piece of malaria DNA with a resistance cassette.
When exposed to antibiotics, due to the resistance marker,
only the bacterial cells that have performed this reaction correctly will survive.
We now need to replace the bacterial resistance genes for a new malaria specific cassette
that will allow us to eventually select for successful vectors in the Malaria parasite.
For this, we first extract the modified DNA – known as plasmids - from the selected bacteria.
We then perform a so-called gateway reaction, to swap one cassette for another.
The modified plasmid is put back into bacteria using electroporation again and then plated up.
Once the bacteria have grown on the plates we pick the colonies and culture them overnight.
As a first quality control step we run another PCR reaction to confirm that the genomic fragment is correctly modified.
We then run the products on a gel and record the results for our sample tracking database.
Once we know the DNA modification has worked the bacteria are stored in freezers.
The bacteria now contain the finals vectors that are ready to modify the genome of Malaria parasites.
We make these available to a worldwide community of scientists through our website
and also use them for large scale experiments of our own.
Working at scale is very different from performing a few individual reactions in the lab,
even at the relatively modest scale of the small Malaria genome.
Procedures need to be highly optimised and standardized and you need to think about tracking the process through the lab.
Good communication between team members is very important.
When you produce at scale any mistakes made also quickly multiply and may cost a lot of time and money to rectify.
I joined the project at an early stage when it wasn't a scaled-up pipeline yet;
it was great to see the progress from a few bespoke constructs to a sizeable scale.
We recently published an important piece of work that probes half of the genome
for the role of genes and growth of the parasite in the blood and it's great to see all that work, culminating in a new scientific discovery.