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Engineering the probiotic Nissle 1917 for oscillatory colorectal therapy I. A. Dragan*, T. Marti* , J. Korzeniowski *

* UNILausanne, Faculty of Fundamental Microbiology - UNIL, Lausanne

Abstract- Colorectal cancer (CRC) incidence and demonstrated (Gao, Zhou, Su, & Huang, 2017; mortality have increased over the past decades, and Huang et al., 2020). The main mechanism through CRC is now the third-most common cancer which azurin acts appears to be by stabilising p53, worldwide. Whilst many new therapies are being leading to its accumulation in the cell, thereby explored, the standards remain chemotherapy, inducing apoptosis (Yamada et al., 2009). radiotherapy and surgery. Recent studies suggest that chronotherapy can reduce side effects without Based on these aspects of cancer research, we loss in efficiency. We aim to use chronotherapy as propose to engineer Escherichia coli Nissle 1917 the basis of our project by introducing a Δclb, a probiotic strain shown to preferentially target repressilator into the probiotic E. coli Nissle 1917, and colonise tumours present in mice (Stritzker et with which we intend to deliver the anticancer al., 2007) to produce azurin at time specific protein azurin in an oscillatory manner. We aim to intervals. To be able to obtain the production and modify the repressilator to produce azurin and to secretion of azurin in an oscillatory manner, we test the full system on Caco-2 cells, our chosen in manipulated the repressilator system described by vitro model. So far, our goals have been met with Potvin-Trottier et al., which has been shown to retain limited success. Despite this, we intend to provide its oscillations once in the gut of mice (Riglar et al., proof of concept of our system within the deadline of 2019). this year’s iGEM competition. Index terms- CRC, iGEM, azurin, probiotics, II. MATERIALS AND METHODS repressilator A. Strains & All strains and the corresponding plasmids used for I. INTRODUCTION the repressilator can be found in the Appendix. Colorectal cancer (CRC) is a growing concern, Nissle 1917 Δclb contains a mutation that removes affecting more and more patients each year. It is the its genotoxicity towards mammalian cells (Olier et third most common cancer, and accounts for a little al., 2012). The pLPT41 is used as a PtetO1 under 10% of all new cancer diagnoses every year molecular sponge to stabilise the oscillations (Kuipers et al., 2015). The most typical treatments generated by the repressilator plasmids. The are surgery, followed by radiotherapy. pLPT119 plasmid is the repressilator plasmid, Chemotherapy is also often recommended alongside containing CFP and mVenus, with no degradation the surgical removal of tumours, in order to prevent tags and so can be used in Nissle 1917. Further reoccurrences (Kuipers et al., 2015). However, these information can be found in the Appendix. therapies are taxing on the patient and often lead to multiple side effects. Due to this, different avenues Those used for protein purification are in Table 2 in of treatment are being explored. One of them is the Appendix. The azurin sequence was taken from chronotherapy, wherein the effect of an anticancer the genome of P. aeruginosa PAO1 and codon drug can vary depending on the timing of its optimized for E. coli ATCC8739 using Genome administration (Eriguchi et al., 2003; Lévi, 2006). It Calligrapher. The azurin(ETH) sequence refers to has been found to be effective when using 5- the export signaling peptide truncated azurin fluoruracil, a common chemotherapeutic drug for sequence used by the 2017 ETH Zurich iGEM team CRC (Mormont & Levi, 2003). (part BBa_K2500001). pelB is a secretion tag, which has been shown to lead to secretion of azurin Another advance in the domain of cancer therapy (Zhang et al., 2012). has been the proposed use of bacterial anticancer peptides to replace more commonly used B. Detection of fluorescence chemotherapeutic molecules (Chakrabarty, For the detection of fluorescence, we adapted the Bernardes, & Fialho, 2014; Thundimadathil, 2012). flask experiment protocol from Potvin-Trottier et al., One such peptide is azurin, a blue copper protein 2016 to be used with a plate reader. Overnight involved in electron transfer during denitrification in cultures of the strains carrying the repressilator Pseudomonas aeruginosa (van de KAMP et al., plasmids were done in 5 mL imagining medium (see 1990). Azurin has become popular as its specificity Appendix) with either isopropyl β-d-1- for cancer cells and cytotoxic activity have been thiogalactopyranoside (IPTG) or

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anhydrotetracycline (aTc) to synchronise the the PLtetO1 of PLPT41, and tested the construct with repressilator system of the cell population at 37°C pLPT119 in Nissle 1917. The measurement of under shaking. The overnight cultures were then mKate2 and CFP do not show the expected diluted to OD600 0.05 in 96-well plates to measure oscillatory behaviour (Figure 3a). We then tested fluorescence. The cells were then diluted every hour the repressilator with the pLPT41-pelB-azurin to keep them in exponential phase to be able to construct and obtained results similar to those with visualise the oscillations. Fluorescence was FIGURE 1: measured in a plate reader using the wavelengths Repressilator and detailed in Table 1. reporter/sponge plasmids. Figure from Table 1: Excitation and emission wavelengths of Potvin-Trottier et al., fluorescent reporter genes 2016

Reporter Excitation Emission mKate2 588nm 633nm mVenus 515nm 527nm CFP 456nm 480nm

C. Protein purification We inoculated 5 mL LB + Amp100 with the strain of interest and let grow overnight at 37°C, shaking. The next day we diluted 1:100 the overnight culture into 10 mL LB + Amp100 and let incubate for 4-5 h at 20°C before inducing protein expression with 0.2% L-arabinose and incubating for 20 hours. The following steps are all done at 4°C. We then harvested 1mL of cells by centrifugation 2 minutes at max speed (20’000g). The cells are then washed FIGURE 2: Fluorescence measurements of mVenus (green) and CFP in 200uL lysis buffer (see Appendix) and sonicated (blue) in Nissle 1917 and DHL708. Measurements were taken every hour for 20 seconds in 1 second intervals at 10% over 11h and kept at an OD600 0.2-0.3. 2a and 2c were synchronised amplitude with the MS73 probe. Once sonicated, the overnight with IPTG, 2b and 2d with aTc. cells are fractionated by centrifuging for 10 minutes at max speed. The supernatant was taken and mixed with 50uL HisMag Sepharose magnetic beads. From this we recovered the flow-through fraction (FT) and then washed the beads with 200uL Lysis buffer before eluting our protein with 50uL Elution buffer (see Appendix). The samples were then mixed with 2X loading buffer (NuPAGE™ LDS Sample Buffer, ThermoFisher), incubated at 95°C for 5 minutes and then loaded onto a 15% acrylamide-bis tris-glycine gel which ran for 30 minutes at 200V. For visualisation we stained with Coomassie Blue for 30 minutes and destained with 50% methanol & 10% acetic acid for 2h.

III. RESULTS AND FINDINGS FIGURE 3: Fluorescence measurements of repressilator constructs over 10h: CFP in blue, mKate2 in red and mVenus in green. OD600 kept between 0.2-0.3. All A. Recreating the repressilator constructs in Nissle 1917 and synchronised overnight with aTc. 3a shows pLPT119 First, we successfully reproduced the oscillation + pLPT41-mKate2, 3b pLPT119 + pLPT41 and 3c pLPT119 + pLPT41-pelB- experiment in the DHL708 strain using pLPT119 azurin. with pLPT41 (Potvin-Trottier et al., 2016, Figure pLPT119+pLPT41 (Figure 3b, 3c). 1). We introduced the repressilator system in Nissle 1917 Δclb and obtained results similar to those with C. Azurin purification DHL708 (Figure 2). Our third goal was to produce azurin from the protein expression strain E. coli BL21. However, we B. Modifying the repressilator were not able to transform this strain with the four Next, we inserted azurin into pLPT41 under the azurin expression plasmids and therefore performed PLtetO1 , so that its expression would the purification of azurin using E. coli Nissle 1917 coincide with that of mVenus on pLPT119. To test Δclb instead. None of the purifications were this co-expression, we first inserted mKate2 under

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successful as no bands at expected size of 16kDa Link to protocols, further results and sequences: were clearly visible (see Appendix.) https://drive.google.com/drive/folders/1utQ4W5X_ HZJgLIhpx_ihHxJzcFL4Rhk4?usp=sharing IV. DISCUSSION As we started our experiments, we soon realised ACKNOWLEDGMENTS that, in contrast to our expectations, we observed a We would like to thank our supervisors, our P.I. constant level of fluorescence for CFP. After Yolanda Schaerli for their guidance throughout our inspection of the plasmid sequence, we noticed that project, as well as Michael Taschner and Roberto this gene is in fact under the control of a constitutive Jareth Vazquez Nunez for their invaluable help with promoter in pLPT119 and so its expression follows the protein purification. the OD600 of our cultures (see Appendix). Despite this, mVenus appeared to be acting in the way we expected. However, the fluorescence measurements REFERENCES Chakrabarty, A. M., Bernardes, N., & Fialho, A. M. (2014). of mKate2 did not act in the expected way originally Bacterial proteins and peptides in cancer therapy: Today and – we believe this is due to its low expression and tomorrow. Bioengineered, 5(4), 234–242. detection as we first kept the OD600 of our cells https://doi.org/10.4161/bioe.29266 around 0.2. Eriguchi, M., Levi, F., Hisa, T., Yanagie, H., Nonaka, Y., & Takeda, Y. (2003). Chronotherapy for cancer. Biomedicine and Pharmacotherapy, 57(SUPPL. 1), 92–95. Modifying the repressilator also proved to be a https://doi.org/10.1016/j.biopha.2003.08.012 challenge, as the size and repetitive sequences of the Gao, M., Zhou, J., Su, Z., & Huang, Y. (2017). Bacterial pLPT119 plasmid complicated its amplification by cupredoxin azurin hijacks cellular signaling networks: Protein– PCR. This difficulty is why we decided to insert protein interactions and cancer therapy. Protein Science, 26(12), 2334–2341. https://doi.org/10.1002/pro.3310 azurin and mKate2 into pLPT41. 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Nature, 538(7626), 514–517. https://doi.org/10.1038/nature19841 V. CONCLUSION AND OUTLOOK Riglar, D. T., Richmond, D. L., Potvin-Trottier, L., Verdegaal, Despite the fact that we encountered multiple A. A., Naydich, A. D., Bakshi, S., … Silver, P. A. (2019). Bacterial variability in the mammalian gut captured by a single- challenges in the implementation of our project, cell synthetic oscillator. Nature Communications, 10(1), 1–12. most notably with the production and purification of https://doi.org/10.1038/s41467-019-12638-z azurin, moving forward we intend to provide proof Stritzker, J., Weibel, S., Hill, P. J., Oelschlaeger, T. A., Goebel, of concept by testing the cytotoxicity of our W., & Szalay, A. A. (2007). Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli engineered Nissle 1917 on Caco-2 cells, our chosen Nissle 1917 in live mice. International Journal of Medical in vitro model. 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8(10), 2947–2958. https://doi.org/10.1158/1535-7163.MCT-09- 0444 Zhang, Y., Zhang, Y., Xia, L., Zhang, X., Ding, X., Yan, F., & Wu, F. (2012). Escherichia coli Nissle 1917 targets and restrains mouse b16 melanoma and 4T1 breast tumors through expression of azurin protein. Applied and Environmental Microbiology, 78(21), 7603–7610. https://doi.org/10.1128/AEM.01390-12

AUTHORS First Author – Ilinca A. Dragan, Bsc Biology, University of Lausanne, [email protected].

Second Author – Thierry Marti, Msc Food Science, University of Lausanne

Third Author – Jakub Korzeniowski, Bsc Biology (ongoing), Faculty of Biology & Medicine, University of Lausanne

Correspondence Author – Ilinca A. Dragan, [email protected].

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