Malaria continues to be a leading case of mortality worldwide. It is estimated that the parasitic disease, carried by infected mosquitoes, spread to over 200 million cases in 2010, resulting in 660,000 deaths.
No vaccine yet
Unfortunately there’s no vaccine yet! For the time being, treatment relies heavily on the use of a limited number of anti-malarial drugs. However, the pathogen – Plasmodium falciparum – is increasingly showing signs of resistance, making the development of new and more effective drugs absolutely critical.
Standard cloning procedure not appropriate
Scientists know genetic approaches are vital to solve this challenge. However, they’ve stumbled upon technical issues using traditional cloning approaches, as the process of constructing the plasmid vectors needed for these studies is very inefficient. This imposes a significant barrier to any genetic manipulations of the parasite.
“For unknown reasons, standard restriction/ligation cloning generate problems with P. falciparum vectors. These problems usually take the form of deletions or rearrangements of the high AT content regions that comprise the 5′ and 3′ UTRs (untranslated regions) necessary for protein expression in the parasite”, explained Jeffrey Wagner, from the Department of Biological Engineering at MIT.
Several aspects of the parasite’s biology contribute to this problem:
- The parasite’s genome is extremely AT-rich (80-90%) with extended regions of low complexity sequence,
- regulatory 5′ and 3′ UTR sequences are poorly defined in P. falciparum, complicating transgene expression and
- the mean coding sequence (CDS) length in P. falciparum (excluding introns) is a long 2.3 kb, nearly twice that of many model organisms
Introducing Gibson Assembly!
To solve this problem, Wagner and colleagues developed a way to apply Gibson Assembly, a new cloning technique, to the parasite’s genome. According to the MIT researcher, this was achieved by creating a family of vectors which could be used not only with standard restriction/ligation-based cloning methods, but more importantly were adapted for Gibson Assembly to generate constructs for use in the parasite.
“There are still instances of plasmid deletions/rearrangements that come up, but they appear to be lessened by using Gibson when compared to restriction/ligation cloning alone”, said Wagner. This method allows to join multiple DNA fragments in a single reaction.
Although it’s not really known why Gibson Assembly is so successful at overcoming the problems, Wagner speculated that it has to do with the much larger “sticky ends” generated with Gibson assembly as compared to standard restriction enzymes (however, this has not been tested yet).
Gibson Assembly: Pros and cons
Gibson Assembly is rapidly becoming a popular choice in many labs, partly because it’s incredibly easy to use, with modular vectors that are simple to assemble. If it involves less steps, fewer reagents and can be done quickly, what is there not to like? Well, the technique does require longer primers, which may increase the chances of synthesis errors as well as being slightly more expensive than conventional approaches. Despite this, Wagner says he has adopted Gibson Assembly wholeheartedly! “At this point, I primarily use Gibson cloning for all of my vector construction. I find it less work intensive than standard restriction/ligation cloning and I also have a better success rate with it”, he concluded.
Wagner J, Goldfless S, Ganesan S, Lee M, Fidock D and Niles J (2013) An integrated strategy for efficient vector construction and multi-gene expression in Plasmodium falciparum. Malaria Journal 2013, 12:373.