transcriptional and translational machinery or post-translational modifications within the host. Other limitations include the labour-intensive library preparation and potential screens which can be both expensive and time-consuming. Alternative approaches Positional cloning Positional cloning is another molecular cloning technique for identification of a gene of interest. This method uses exact chromosomal location instead of function to guide gene identification. Because of this, this method focuses on all the genetic material at a chromosomal locus and makes no assumptions about function. In model organisms such as mice or yeast, this method is used more frequently as the information about the position of a gene of interest can be obtained from the sequenced genome. However, this method becomes much more cumbersome when sequence information is not available. In this case, linkage analysis can also be used. Functional cloning on the other hand is more readily used in organisms such as bacterial pathogens that are viable but nonculturable and where sequence data is not available but gene homology or protein function is still of interest. A way to differentiate between functional and positional cloning is to visualize genes as words. Functional cloning is like using a thesaurus to look up words and selecting for new words that have the same meanings (or functions). Positional cloning is more like picking a specific page of a dictionary and then browsing only that page for any words of interest. Computationally determine homology With the advent of sequencing technology becoming cheaper and cheaper, it is now more feasible to sequence an unknown genome and then computationally determine homology instead of screening. This brings the added benefit of being able to screen for multiple genes of interest at the same time and reduces experimental time. It also allows one to avoid labour-intensive cloning procedures as well. However, if this route is taken, there are other biases and hurdles one must consider. By using sequenced data, one is able to screen based on homology alone. A function-based approach thus allows for discovery of novel enzymes whose functions would not have been predicted based on DNA sequence alone. Therefore, while sequencing is less labour-intensive experimentally, it can also lead to missed genes of interest due to differing sequence homology in genes of related function. Gibson assembly Gibson assembly is a quick cloning method that uses three primary enzymes; 5' exonuclease, polymerase and ligase. The exonuclease digests the 5' end of DNA