Team:NUS Singapore/Project

Interactive Points | Codrops

Part 1:
RIOT Sensor


We synthesized a series of RIOT sensors which is repressed by LldR and activated by L-lactate. Among them, Construct 7 and Construct 8 exhibited an increased sensitivity to lactate compared to the original sensors (namely, Construct 1 and 2).

To enhance the specificity of our RIOT system, we inserted Construct 7 in a low-copy plasmid backbone to fine-tune the expression of ALR. This may reduce their growth in the bloodstream and keep them from attacking healthy cells, which do secrete some concentration of lactate.

BACKGROUND:

Due to the ability to deregulate normal cellular energetics, cancer cells are found to undergo aerobic glycolysis, followed by lactic acid fermentation in the cytosol, resulting in the elevated production and accumulation of L-lactate in the surrounding environment (Chen et al., 2007; Vander et al., 2009; Alfarouk et al., 2015). In our group’s effort to specifically distinguish cancer cells from normal cells, we designed a collection of RIOT sensors which are responsive to L-lactate concentrations.

With specialised promoters from our collaborator, team
2015 ETH_Zurich , our RIOT sensors contain an alanine racemase (ALR) gene under the control of a lactate-sensitive promoter (Figure 1). ALR is an enzyme that catalyzes the reaction of L-alanine ⇌ D-alanine. D-alanine is an essential amino acid that is required for bacterial cell survival (Walsh, 1989). One of our goals is to make the RIOT sensor activated in the presence of high lactate concentration, which enhance the survival of the bacteria so that they are able to attack the cancer cells more efficiently while leaving normal cells unharmed.

Fig 1. Mechanism of RIOT sensor. (A) In the absence of lactate, lldR binds to two operators in the promoter region and inhibit the expression of Alanine Racemase (ALR). D-alanine is an essential molecule for the cross-linkage of the peptidoglycan layer in bacterial cell wall. Therefore, without ALR, a lack of D-alanine in auxotrophic mutant bacterial inhibits cell division and cell growth (Aguilera, 2008). (B) In the presence of lactate, lactate binds lldR, preventing its binding to bind the operators. Consequently, ALR is expressed and catalyzes the conversion of L-alanine to D-alanine which is required for the survival of bacteria.

Part 2:
RIOT Transponder


Here, we use the CD44v6 antibody, which is conjugated to a bacterial peptide called hasA. hasA is part of the Has operon system, which when bound to hasR (found on the surface of the bacterial cells) leads to a cascade of reactions within.

The Has operon is another critical sensor for the RIOTsystem. The proteins involved in the operon are hasA, hasR, hasS and hasI. hasA is an extracellular haemophore and it binds to bacterial cells expressing hasR, the hasA receptor. This binding causes a conformational change in hasS (an anti-sigma factor), which then releases hasI, a sigma factor. hasI then binds to its specific promoter (labelled as pHas) to trigger expression of genes under pHas. In the RIOT system, the gene expressed is luxR, which is required to activate the RIOT suppressor.

Part III:
RIOT Responder-Invader

LuxR is under the control of pHas, which get activated via transponder circuit. The production LuxR will then induce the expression of LuxI, invasin, LLO (listeriolysin O) and GFP respectively. There is already a basal level of AHL in the bacteria. Upon LuxI expression, more AHL is produced, thus amplifying the loop of this positive-feedback mechanism.

Invasin helps the bacteria to get into cell endosome while LLO which is a pore-forming toxin will allow the bacteria to escape out of the endosome into cytosol. We use GFP as a marker to locate the bacteria and signal the successful activation of circuit.

BACKGROUND:

Invasin is derived from the bacteria Yersinia pestis which allows selective invasion of cells that express β1-integrins. Listeriolysin O (LLO) on the other hand, is expressed in Listeria monocytogenes and allows the bacteria to break out of the endosome after entry into mammalian cells. LLO is activated in the low pH enviroment (pH 5.5) of the endosome. Then, LLO will forms pores in the membrane of the endosome and results in the lysis of the endosome and the escape of the bacterium into the cytoplasm. Combining the traits of these two genes, our engineered bacteria is capable of entering the cytosol of cancer cells to potentially release a cytotoxic drug.

We aim to make a construct that is activated in the presence of a hasS-hasI complex that is formed in the presence of CD44v6 on the surface of cancer cells. The construct makes use of luxR and luxI to activate the production of invasin and listeriolysin O (LLO), as well as a designated drug that will be able to kill off the cancer cells.

The combination of both invasin and LLO allows the bacterium to invade mammalian cells and escape from the endosome into the cytoplasm of the cell. Once in the cytoplasm, the drug released from the bacterium can build up within the cell to result in the destruction of the cell. Combined with RIOT Sensor and RIOT Transponder, the bacterium can potentially target and eliminate cancer cells with minimal side effects.

Initially, small amounts of N-Acyl homoserine lactones (AHLs) produced by our engineered bacteria freely diffuse in and out of the cell. In tumor regions with an expected higher cell density, the concentration of AHL also increases. Past a threshold, LuxR binds to AHL and this LuxR-AHL complex will further activate expression of LuxI, producing more AHL. This creates a positive feedback loop that increases both the concentration of LuxI and of our desired proteins, invasin and LLO.