Team:UNILausanne/Results

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Results Overview

Overview

  • We designed and constructed plasmids for E. coli Nissle 1917 Δclb to produce and secrete the anti-cancer peptide azurin in an oscillatory manner by using a repressilator
  • We demonstrated that the repressilator works in our chosen chassis
  • We demonstrated that the azurin is produced and also secreted
  • We characterized the effect of anti-cancer drugs on colorectal cancerous Caco-2 cells
  • We assembled a kill switch so that our engineered bacteria will not survive outside the patient's gut

Repressilator

Overview

Chronotherapy is a promising novel approach to treat cancer. Studies suggest that administration of anti-cancer drugs at a certain point of the circadian rhythm has a positive impact on the treatment efficiency and can lower side effects. We aim to combine E. coli Nissle 1917 Δclb bacteria‐assisted tumor‐targeted therapy with chronotherapy to explore a new method of fighting cancer. The oscillatory release of the anti-cancer drug is achieved by using a synthetic oscillatory circuit called the “repressilator”.

Key Achievements

Figure 1A: Modelled oscillatory expression pattern of the repressilator with three fluorescent reporter genes under promoters lacl, tetR and cl. 1B: Oscillatory pattern obtained by transforming E. coli DHL708 with BBa_K3482023 (pLPT234) and Part:BBa_K3482024 (pLPT145), which are respectively the repressilator plasmid and the sponge plasmid. The repressilator is based on 3 repressor proteins associated to three fluorescent proteins, which are CFP, mKate2 and mVenus, under the same 3 promoters that we can see on Figure 1A (CFP is under p_tetR, mKate2 under p_lacl and mVenus under p_cl) and the sponge plasmid was used to stabilize the oscillator. The bacteria were diluted to the same OD so the fluorescence would not depend on the quantity of bacteria. All bacteria were synchronized with aTC or IPTG to start oscillations at the same time. A plate reader was used to measure OD and fluorescence for each reporter gene. Data shows the mean and the standard deviation of 3 replicates.
  • We tested an established repressilator system and modified it with our gene of interest
  • We established and optimized a protocol that allowed us to detect oscillations by the repressilator in a plate reader assay

Azurin secretion

Overview

Azurin is a copper-binding redox protein originally found in Pseudomonas aeruginosa. In P. aeruginosa, azurin is exported into the periplasm. Azurin reduces the growth of human breast cancer and melanoma tissue with minimal side-effects. Moreover, it can be produced in E. coli. To make azurin an effective drug it needs to be secreted outside the bacterial cell in the vicinity of the colorectal tumor. Therefore, we used E. coli Nissle 1917 Δclb as our chassis and engineered the azurin gene so it can be secreted.

Key Achievements

  • We added either pelB-5D or NSP4 secretion tags to the azurin and showed that the expression and secretion of azurin by our designed bacteria
  • We inserted the azurin with either peIB-5D or NSP4 secretion tags into the sponge plasmid of the repressilator

MTT assay

Overview

The effectiveness of azurin against cancer cells has only been tested on breast tumors and melanoma up to now. Azurin showed significant effects on these kinds of cancer because it can amplify the effect of p53, an intracellular protein which acts as a pro-apoptosis factor. However, the effects of azurin on colorectal cancer cells are still unknown. The susceptibility of the colorectal cancer cell line called Caco-2 to different anti-cancer drugs was characterized for the first time in the iGEM competition.

Key Achievements

Figure 2A: Caco-2 cell viability (%) when they are incubated with 0.15 % Triton X or Medium or purified azurin [2 μM]. 2B: Plasmids for each color: blue, negative control; red, negative control for the azurin secretion; green, PeIB-5D secretion tag on azurin; purple, NSP4 secretion tag on azurin.
  • We tested different concentrations of anisomycin, cisplatin, doxorubicin, salirasib and TNFa on Caco-2 cells
  • We applied purified azurin on Caco-2 cells, but azurin does not have an effect on the Caco-2 cell viability at the tested concentration (Figure 2A)

Kill switch

Overview

To prevent the survival of the GMOs in the environment and in the patient's body outside the colon, we designed a kill switch. The kill switch prevents undesired survival of our chassis by killing it with a toxin/antitoxin system at two conditions: when the temperature is lower than 37°C (body temperature) and when the phosphate concentration is too high (conditions of the bloodstream). Moreover, the kill switch is intended to act as an addiction module that allows us to maintain our plasmids without the need of antibiotic resistance genes within the patient’s gut.

Key Achievements

  • We assembled a multi-responsive sensor: it reacts on temperature and phosphate concentration
  • we characterized the toxin-antitoxin systems ccdb/ccda and miniColicin E2/IM2 and identified the promoter induction range for differential cell growth inhibition
  • We demonstrated that E. coli Nissle 1917 ΔclB has a sensitive gyrase genotype to the ccdB toxin
  • We characterized our new composite part (BBa_K3482017)
  • We demonstrated that our kill switch system controls cell growth effectively depending on temperature

A big thank you to our sponsors for their valuable support!