Team:NEU CHINA/project design.html

 PROJECT_Design

3.Amplifier and Tuner
    For our purpose, it is vital to select a suitable and easily modified chassis for anti-inflammation. Escherichia coli (E. coli) is a classical and very useful organism for the production of recombinant proteins in synthetic biology. It has become the most popular prokaryotic expression platform [1]. For this reason, there are many protocols and methods for high-yield production of heterologous proteins, such as a vast catalog of expression plasmids, engineered strains and various cultivation strategies. As a well-established protein production system, E. coli has many advantages. (i) It has fast growth kinetics. Under the optimal cultural conditions, its doubling time is only about 20 min [2]. Thus, the high cell density cultures are easily obtained within a short time. According to previous studies, the theoretical density limit of an E. coli liquid culture is about $10^{13}$ viable bacteria/ml [3,4]. (ii) The culture conditions and rich complex media can be made from readily available and inexpensive components. (iii) E. coli stains are easily stimulated to competent cells, which much easily absorb the exogenous DNA via transformation. The exogenous DNA transformation is very high effective, even can be done within 5 min [5]. In this project, we used two E. coli based competent cells, E. coli DH5α and E. coli BL21, to express our anti-inflammation proteins. Meanwhile, we made the E. coli Nissle 1917(EcN) based competent cell to express the inflammation sensing protein. We intended to express recombinant proteins only E. coli Nissle 1917 strain. Why to choose EcN as our chassis is not only because it is one of the E. coli strain, but also it is a clinical determined and non-harmful probiotic bacterium for promoting intestine health [6]. This strain was firstly used as a therapeutic agent for experimental therapy of intestinal bowel disease (IBD) in humans. As one of the clinical approved bacteria strain, EcN lacks virulence factors and is confirmed as the therapeutic safety strain [7]. In addition, EcN with several distinctive features, include microcin synthesis and different iron uptake systems, which offer EcN with the advantages for intestinal colonization [8]. Furthermore, Bruder’s team indicated that the recombinant EcN with no harmful effect in migration, clonal expansion and immune cell activation in immunocompetent host [9]. Therefore, E. coli Nissle 1917 may act an ideal bio-engineered bacterial strain for gastrointestinal related in situ synthesis of inflammation sensing proteins and anti-inflammatory molecules.
PROJECT
    Considering the impact of colonization of exotic microorganisms in the intestine and the goal to accelerate the secretion of IL-10 and myrosinase, we found a gain-tunable transcription amplifier in Pseudomonas syringae to tunable amplify the NO signal.
    The natural gene regulatory architecture in the hrp control network contains the ingredients of a tunable transcriptional amplifier: the activator proteins HrpR and HrpS form a sensitive high-order co-complex, which binds the upstream activator sequence of the hrpL promoter to remodel the closed σ54 -RNAP-hrpL transcription complex to an open one through ATP hydrolysis, while the formation of co-complex can be tuned by the inhibitor HrpV through its interaction with HrpS(Fig. 4) [2].

Overview
DESIGN
    Last year, NEU_China_A creatively proposed the use of anti-inflammatory cytokine, IL-10, to regulate excessive intestinal inflammation in IBD patients and express a natural anti-cancer substance in bacteria to alleviate inflammation and bowel cancer. Unlike traditional medicine therapy, immunotherapy improves the host's immune regulation ability and can largely avoid the problem of drug resistance, relapsing and deterioration for IBD [1]. Unfortunately, this original project left a lot of work to do.
    This year, we extended the project with the intestinal microbiota transplantation by using the Escherichia coli Nissle 1917 (EcN), a kind of probiotics in human gut, as the chassis for “gut firemen”. Last year, the team only came up with this conception; this year, we attempted to transform our plasmid to EcN and characterized the function in EcN.
    Considering the sensitivity of “gut firemen”, we optimized the nitric oxide sensors from the last year’s team and successfully reduced the leakage rate of the sensor with an extra binding sequence of regulator proteins. Besides, we constructed another new nitric oxide sensor and characterized it for a better choice.
     Although the amplifier’s idea had been come up with by NEU_China_A 2018 team, they did not really construct it. In this iGEM year, we found a feasible system in Pseudomonas syringae and designed a tunable-gain amplifier. And we built the Leakage Model of fixed-gain amplifier to explore more about the tunable amplifier.
     Last year, we only confirmed the gene circuit work using the GFP as the reporter gene. For the future protein experiments, this year, we characterized the therapeutic compounds, IL-10 and myrosinese, using immunoblotting method. To confirm the activity of those compounds expressed by E. coli, we tested the activity of IL-10, the immune cytokine.
     For the biosafety, NEU_China_A 2018 did not get the satisfied “kill switch” system due to the serious toxin expression leakage at 37℃, which meant only few bacteria survived at that temperature. This year, with the toxin-antitoxin system-mazEF, we significantly increased cell viability and precisely control the anti-bacterial toxin expression at different temperatures.
      All those works and optimizations contributed to the creation of “gut firemen” that enabled to precisely colonize in the gastrointestinal inflammation region; successfully expressed and secreted anti-inflammatory proteins and put out the fires in guts with few biological hazards and least side-effect.

1.Suitable chassis
2.NO-dependent Inflammatory sensor
     Our bio-engineered bacteria should be enabled to relieve the symptoms of gut inflammation. In order to achieve this purpose, the bacteria should sense the inflammation signals first and trigger the anti-inflammatory proteins expression. Several studies have demonstrated that the nitric oxide (NO) is a critical signal of the gastrointestinal inflammation, and the concentration of NO is positively related to the severity of intestinal inflammation [1]. The average concentration of nitric oxide in the rectum of patients with IBD reaches 5.5 μM, while the normal people reaches 60 nM, it nearly over 100 times differences [2]. Thus, NO is an ideal input signal to reflect the gut inflammation. Last year, we used NsrR-yeaR and NorR-norV based NO sensor plasmids for NO detecting. This year, we would like to design and construct a novel NO sensor system with highly NO sensing specificity. Saraiva’s team found that the ytfE gene expression is significantly induced under the nitrosative stress from the transcriptomic analysis [3]. Furthermore, the ytfE strain is much more sensitive to nitric oxide than the parental E. coli strain [3]. Thus, we used ytfEΔ as a novel sensing system to compare with our previous sensors to demonstrate the most sensitive and specific NO sensor.
    Among two NO sensing plasmids, we chose luciferase as the reporter gene based on its three main advantages: convenient, relatively inexpensive and providing quantitative measurements instantaneously. Its extreme sensitivity allows quantification of even small changes in transcription and the availability of results within minutes of completing our experiment makes it even more appealing. Last year, our prior work indicated that the expression leakage of NsrR-yeaR and NorR-norV sensors. This year, we have figured out several solutions and the results are obvious. For more details, please refer to “Improved Biobricks”.
Figure 4. The composition of tunable biological amplifier. Transcription input can be amplified through the amplifier.
     In order to increase the expression of anti-inflammatory protein expression, we designed the tunable biological amplifier. Briefly, the tunable biological amplifier (Fig. 4) comprises three modular parts - the input, the output and a gain-tuning input. The device can continuously process the input transcriptional signal with an externally tunable gain (the amplification ratio of the changes in output to input) control [1].
REFERENCE

Figure 3. Engineered bacteria with inflammation sensors. PyeaR, a promoter which is sensitive to NO. PytfE, a promoter which is sensitive to NO. RBS, ribosome binding site. NsrR, regulator protein which can bind to NO. Luciferase, reporter gene. Terminator B0010/B0012, double terminator.
    In this project, we used our engineered bacteria strain to express anti-inflammatory proteins to relieve IBD symptoms. As a complex chronic disorder, IBD can be induced by various factors which we have mentioned before. Among these factors, immunity imbalance acts as a critical inducer and over-activated immune cells produce a large number of cytokines that lead to intestinal layer destruction and inflammation. However, cytokines production is extremely regulated in immunocompetent host. Among these cytokines, interleukin-10 (IL-10) plays a central role in downregulating inflammatory cascades and maintains the intestinal layer functioning [1]. Furthermore, IL-10 has been determined in preventing cell accumulation, which consequently alleviates IBD. However, the administration of purified IL-10 protein via oral and intravenous routes is not practical. Therefore, we used genetically modified E. coli strain to express IL-10 protein to relieve chronic inflammation [2].
4.IBD ameliorating proteins
[1]Catalan-Serra I, Brenna Ø. Immunotherapy in inflammatory bowel disease: Novel and emerging treatments. Hum Vaccin Immunother. 2018;14(11):2597–2611. doi:10.1080/21645515.2018.1461297
Our core design consists of the following:
REFERENCE

[1]Rosano, G.L. and E.A. Ceccarelli, Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol, 2014. 5: p. 172.
[2]Sezonov, G., D. Joseleau-Petit, and R. D'Ari, Escherichia coli physiology in Luria-Bertani broth. J Bacteriol, 2007. 189(23): p. 8746-9.
[3]Lee, S.Y., High cell-density culture of Escherichia coli. Trends Biotechnol, 1996. 14(3): p. 98-105.
[4]Shiloach, J. and R. Fass, Growing E. coli to high cell density--a historical perspective on method development. Biotechnol Adv, 2005. 23(5): p. 345-57.
[5]Pope, B. and H.M. Kent, High efficiency 5 min transformation of Escherichia coli. Nucleic Acids Res, 1996. 24(3): p. 536-7.
[6]Schultz, M., Clinical use of E. coli Nissle 1917 in inflammatory bowel disease. Inflamm Bowel Dis, 2008. 14(7): p. 1012-8.
[7]Hancock, V., M. Dahl, and P. Klemm, Probiotic Escherichia coli strain Nissle 1917 outcompetes intestinal pathogens during biofilm formation. J Med Microbiol, 2010. 59(Pt 4): p. 392-9.
[8]Sassone-Corsi, M., et al., Microcins mediate competition among Enterobacteriaceae in the inflamed gut. Nature, 2016. 540(7632): p. 280-283.
[9]Westendorf, A.M., et al., Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules. FEMS Immunol Med Microbiol, 2005. 43(3): p. 373-84.

Figure 2. Pellets of bacteria transformed with constructed NO sensor plasmid after 6hrs inducted at 37 ℃ from iGEM 2018NEU_ChinaA. From left to right: control, 0.5mM IPTG without SNP, 1mM IPTG without SNP, 0.5mM IPTG with 100μM SNP, 1mM IPTG with 100μM SNP. 100μM Sodium Nitroprusside Dihydrate (SNP) aqueous solution could continuously release NO and its final concentration is stable at about 5. 5μM.The third and fourth pellets demonstrate the serious leakage of the previous NO sensors.
    In the ytfE and yeaR based NO sensing plasmids, NsrR protein act as the negative regulator for transcription via binding to the yeaR and ytfE promoters (PyeaR and PytfE). The repression would be eliminated by nitric oxide strongly attaches to the NsrR protein and lead to detach from promoters [4].
REFERENCE

[1]Rachmilewitz, D., et al., Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease. Gut, 1995. 36(5): p. 718-23.
[2]Ljung, T., et al., Rectal nitric oxide assessment in children with Crohn disease and ulcerative colitis. Indicator of ileocaecal and colorectal affection. Scand J Gastroenterol, 2001. 36(10): p. 1073-6.
[3]Justino, M.C., et al., Escherichia coli YtfE is a di-iron protein with an important function in assembly of iron-sulphur clusters. FEMS Microbiol Lett, 2006. 257(2): p. 278-84.
[4]Karlinsey, J.E., et al., The NsrR regulon in nitrosative stress resistance of Salmonella enterica serovar Typhimurium. Mol Microbiol, 2012. 85(6): p. 1179-93.

Figure 5. The architecture of the tunable-gain amplifier. A tunable-gain transcriptional amplifier is designed on the transcriptional regulators HrpR (R), HrpS (S) and HrpV (V) in the hrp (hypersensitive response and pathogenicity) gene regulatory module from P. syringae.
    With such a tunable-gain amplifier, our engineering bacterium can be under a precise control. Our IBD patients can eat some arabinose to release the anti-inflammatory effect of our bacterium when the clinical symptoms begin to relieve.
    Using a multilayered amplifier cascade, built from cascaded orthogonal other amplifiers, to sequentially amplify the transcriptional input signal flow is a considerable choice when the amplification gain is not distinct as expected induction [3].

REFERENCE

[1]Jovanovic, M., et al., Regulation of the co-evolved HrpR and HrpS AAA+ proteins required for Pseudomonas syringae pathogenicity. Nat Commun, 2011. 2: p. 177.
[2]Levskaya, A., et al., Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature, 2009. 461(7266): p. 997-1001.
[3]Steidler, L., et al., Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science, 2000. 289(5483): p. 1352-5.

Interleukin-10
Figure 7. The effect of our anti-inflammatory E. coli. Excessive inflammation happens when immune cells such as effector T cells and macrophages keep accumulating. But Interleukin-10 can stop this trend [3].
Myrosinase
    Besides the IL-10 protein, we are also interested in integrating another beneficial protein, myrosinase. Although IBD is considered as a chronic inflammation, the long term and un-suppressed IBD is highly related with the colorectal cancer incidence [4]. Chemoprevention refers to a method using pharmacological or natural agents for therapeutic purpose, such as anticancer treatment. There are several chemoprevention agents have been determined with effective anticancer effects. The glucosinolate and myrosinase are widely spread in cruciferous plants; myrosinase is an enzyme that mediates the conversion of glucosinolate to sulforaphane (SFN), which has been reported for suppression of tumorigenesis in solid tumors [5]. Sulforaphane can mediate cancer cell cycle arrest (G2/M) which kills the cancer cells, and it also can also activate the Nrf2-ARE pathway that releases the oxidative stress for anticancer effects [6]. However, mammals do not express myrosinase and the conversion of glucosinolate by the host microbiota shows inadequate to reach anticancer effects [7]. In that case, chemoprevention which means that introducing cruciferous plants diet along with myrosinase expressing strain can be feasible to do a better anticancer strategy. In our project, we used one bacterial chassis to express two beneficial proteins only under the inflammation induction.
Figure 8. Diet-mediated tumor chemoprevention and inflammation relief. Diet-mediated tumor chemoprevention and inflammation relief. Glucosinolate is widely spread in cruciferous and so do myrosinase, an enzyme mediating the conversion of glucosinolate to sulforaphane. Sulforaphane (SFN) has been reported to indicate for the prevention and suppression of tumorigenesis in solid tumors. It can activate the phase 2 and Nrf2-ARE pathway to kill cancer cells and to upregulate HO-1 and SOD to release oxidative stress which consequently ameliorates IBD [4].
REFERENCE

[1]Steidler, L., et al., Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science, 2000. 289(5483): p. 1352-5.
[2]Steidler, L., et al., Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat Biotechnol, 2003. 21(7): p. 785-9.
[3]Liang YE and Huang Z. IL-10 Signalling in Macrophage during Autoimmunity. Austin Biol. 2016; 1(1): 1004.
[4]Axelrad, J.E., S. Lichtiger, and V. Yajnik, Inflammatory bowel disease and cancer: The role of inflammation, immunosuppression, and cancer treatment. World J Gastroenterol, 2016. 22(20): p. 4794-801.
[5]Lin, L.C., et al., Sulforaphane potentiates the efficacy of imatinib against chronic leukemia cancer stem cells through enhanced abrogation of Wnt/beta-catenin function. J Agric Food Chem, 2012. 60(28): p. 7031-9.
[6]Zhao, H.D., et al., Sulforaphane protects liver injury induced by intestinal ischemia reperfusion through Nrf2-ARE pathway. World J Gastroenterol, 2010. 16(24): p. 3002-10.
[7]Ho, C.L., et al., Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention. Nat Biomed Eng, 2018. 2(1): p. 27-37.

5.  Kill switch
    Last year, the cold shock kill switch we designed was based on the toxin-antitoxin system-mazEF, a natural toxin-antitoxin system found in E. coli. The MazF is a stable toxic protein, while MazE has the feature of instability [1]. When environmental stress represses bacteria and inhibits the expression of mazEF, the unstable antitoxin protein, MazE, is antecedent to be degraded，and the relative content of the stabilized toxic protein, MazF, increases to a certain degree, which is the direct cause for bacterial death [2, 3]. The translation of the mRNA mediated by the cold-acting promoter PcspA can only be activated at 16℃ [4]. Because of the limitation of time, last year we only transformed the plasmid with the mazF gene induced by cold-acting promoter PcspA to prevent engineered bacteria from leaking into the environment. This year, we measured the survival rate of bacteria transformed with the last year constructed plasmid. However, results showed that the survival rates were low at both 16℃ and 37℃. Therefore, we suspected the high toxicity of MazF directly results in bacterial death. We co-transformed the two plasmids, PT7-mazE and PcspA-mazF into E. coli BL21. After repetitive experiments and constant measurement, the survival rate of mazE and mazF transformed strain reached to 88.06% at 37 ℃. The result suggested that the survival rate of engineered bacteria transformed with double plasmids had substantially increased, which indicated the introduction of MazE is of great significance for promoting the kill switch function.
Figure 9. Schematic Design of the Kill Switch. PcspA, a cold-acting promoter which can only be activated at 16℃.PT7, the gene downstream of this promoter will be transcribed when there is T7 RNA polymerase. MazF, a stable toxic protein, under the control of the T7 promoter, which can kill the bacteria at low temperature. MazE, an unstable antitoxic protein, which prevents MazF from killing the bacteria at 37 ℃.
REFERENCE

[1]Aizenman, E., H. Engelberg-Kulka, and G. Glaser, An Escherichia coli chromosomal "addiction module" regulated by guanosine [corrected] 3',5'-bispyrophosphate: a model for programmed bacterial cell death. Proc Natl Acad Sci U S A, 1996. 93(12): p. 6059-63.
[2]Pandey, D.P. and K. Gerdes, Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes. Nucleic Acids Res, 2005. 33(3): p. 966-76.
[3]Amitai, S., Y. Yassin, and H. Engelberg-Kulka, MazF-mediated cell death in Escherichia coli: a point of no return. J Bacteriol, 2004. 186(24): p. 8295-300.
[4]Stirling, F., et al., Rational Design of Evolutionarily Stable Microbial Kill Switches. Mol Cell, 2018. 72(2): p. 395.

Figure 1. The diagram of our project, the “gut firemen” bacteria.
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NEU-CHINA
Figure 6. The architecture of the tunable-gain amplifier. A tunable-gain transcriptional amplifier is designed on the basis of the transcriptional regulators HrpR (R), HrpS (S) and HrpV (V) in the hrp (hypersensitive response and pathogenicity) gene regulatory module from plant pathogen P. syringae.
    Based on that, we connect our NO sensor part, the improved PyeaR, to the input of the fixed-gain amplifier with the IL-10 and myrosinase as the output. For the reducing secretion when the inflammation is not so serious, we add a tuner for the amplifier with the inhibitor protein, HrpV (Fig. 6).