Part:BBa_K3419000
Contents
Aspartate-semialdehyde Dehyodrogenase (Asd)
Usage and Biology
Note: All work on this basic part was done virtually through literature research due to COVID-19 restrictions.
The presence of an engineered plasmid adds a burden to the cell’s metabolic processes, causing the bacteria that carry the plasmid to be selected against. Since our treatment is meant for the human body, we do not want to use traditional methods for selection - such as antibiotic resistance - to ensure that our plasmid is propagated. This is because the human body cannot handle large amounts of antibiotics, especially given that patients are expected to undergo other cancer treatments combined with our therapy. For our therapeutic bacteria, we examined knocking out the gene for aspartate-semialdehyde dehydrogenase (Asd) and placing a copy of it on our desired plasmid. Asd is an essential gene in many bacteria, including E. coli, that must be retained in order to survive in environments not specifically enriched with diaminopimelic acid (DAP), the crucial product of Asd used in forming cell walls. Using an Asd obligate ensures that successive generations of E. coli include our therapeutic system and other components of our plasmid without the need for antibiotics.
Older studies have successfully used E. coli with faulty Asd genes to create “antibiotic marker-free plasmids” [1]. Since DAP is critical for the bacterial cell wall, the absence of the Asd gene leads to the lysis of bacterial cells. In one study, when plating the transformed bacteria on agar with DAP, the plasmid retention rate is found to range from 80-100% depending on the dilution factor [1].
Mechanism behind Lambda Red Gene knockout:
The system that would be used for Asd gene knockout is lambda red recombinase-mediated homologous recombination [2]. This mechanism for gene knockout required linear DNA fragments carrying kan‐kil genes and homologous extensions to the targeted locus. An Asd expression “cassette” is inserted with nirB promoter into a eukaryotic vector, allowing for the replacement of Asd by lambda red.
The template plasmid containing the replacement gene sequence has primer sites on either end of the gene allowing the oligonucleotides to anneal at these primer sites. PCR using oligonucleotide sequences combined with the gene sequence as a template creates linear dsDNA.
The dsDNA PCR product is acted upon by an exoenzyme: an exonuclease that removes nucleotides from one strand of the dsDNA PCR product. This allows beta proteins to attach to the “middle” of the dsDNA, thereby ensuring that the dsDNA strand does not get deleted by exoenzymes.
The result is a dsDNA PCR product whose “middle” is the antibiotic gene that replaces the Asd gene, and “ends” are “overhangs” that allow the PCR product to be inserted into the bacterial chromosome.
References:
[1] Shi, X., Wang, J. (2015). Engineering and characterization of a symbiotic selection-marker-free vector-host system for therapeutic plasmid production. Molecular Medicine Reports, 12(3), 4669-4677. doi:10.3892/mmr.2015.3945
[2] NanoBio: Protocol for gene knockout. (n.d.). Retrieved October 17, 2020, from https://openwetware.org/wiki/NanoBio:_Protocol_for_gene_knockout
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