Trichosanthin is a potent anti-tumor protein that inactivates eukaryotic 60s ribosomal subunits. It does so by catalyzing the endohydrolysis of the N-glycosidic bond at one specific adenosine on the 28S rRNA [1]. This part also features a translocase tag to export trichosanthin out of the E. coli to improve efficacy, as shown by Figure 1.
Figure 1. Trichosanthin is an inhibitor of protein synthesis that acts on mammalian ribosomes. Upon release into the cell, it exhibits RNA N-glycosidase activity and depurinates the 28S rRNA of the 60S eukaryotic ribosomal subunit. This will achieve irreversible inactivation of ribosome activity and inhibit tumor cell function.
Aspartate-semialdehyde dehydrogenase (Asd) is an essential gene in E. coli that we leverage in order to ensure the retention of our plasmid in bacteria cells. It plays an important role in the biosynthesis of amino acids and bacterial cell wall formation [2]. By knocking out the Asd gene in the bacterial chromosome and then reintroducing it through our plasmid, we ensure that only cells that retain the plasmid will survive.
LldR is a regulatory protein that acts as a part of the lldPRD operon of E. coli. When bound to lactate, LldR allows RNA Polymerase to bind and transcribe the downstream gene. When not bound to lactate, LldR represses transcription [3].
mCardinal is a red fluorescent protein with a peak excitation wavelength of 604nm and a peak emission wavelength of 659nm. Our system expresses mCardinal under a constitutive promoter so that the location of our bacteria, and thus the tumor, can be tracked throughout the body [4].
Our kill switch system consists of holin, a protein that creates holes in the bacterial cell membrane and causes cell lysis, and antiholin, a protein that binds to holin and inactivates it [5]. Cell survival depends on the relative concentrations of holin and antiholin. As shown in Figure 2, we express antiholin under a lactate-inducible promoter so that it is only expressed in the high-lactate tumor environment, preventing the bacteria from surviving outside of the tumor.
Figure 2. Diagram depicting the mechanism behind this lactate-inducible kill switch, which will respond to the high lactate levels in cancer cells due to increased aerobic glycolysis.
[1] SIB Swiss Institute of Bioinformatics. (2020, June 17). Ribosome-inactivating protein alpha-trichosanthin. Retrieved October 05, 2020, from https://www.uniprot.org/uniprot/P09989
[2] Aspartate-semialdehyde dehydrogenase. (n.d.). Retrieved October 05, 2020, from https://www.ebi.ac.uk/interpro/entry/InterPro/IPR012080/
[3] Khartchenko, A. (n.d.). BBa_K1847004. Retrieved October 05, 2020, from http://parts.igem.org/Part:BBa_K1847004
[4] Lambert, T. (n.d.). MCardinal at FPbase. Retrieved October 05, 2020, from https://www.fpbase.org/protein/mcardinal/
[5] Source: Savva, C. G., Dewey, J. S., Deaton, J., White, R. L., Struck, D. K., Holzenburg, A., & Young, R. (2008). The holin of bacteriophage lambda forms rings with large diameter. Molecular microbiology, 69(4), 784-793.