Bioluminescent Bacteria

RELEASED Project Lux Aeterna Released for Research on 09.27.2025 By the Global Polaris Coalition Department of Biomedicine Subsector of Microbiology and Genetics ​ Project Manager: ISO-OMICRON Chief Scientific Officer: ISO-OMICRON​ Participating Researchers: ISO-AQUILA, MESO-GIANI, ISO-YAMAL, MESO-BINARY, EXO-MIT, MESO-ECO, INT-RABI, MESO-AZRA, EXO-NATSU, EXO-ACE, ALPHA-TAU, POLY-TAI, NEO-REDDY, POLY-SUPER, POLY-MALIKAH,POLY-TORI, Collation: 8 (pg.) Document ID: BM032025 Document Name: Refined Analytics on In Vitro Genetic Modification of Escherichia Coli K-12 with the Lux Operon Refined Analytics on In Vitro Genetic Modification of Escherichia Coli K-12 with the Lux Operon ​ Abstract The objective of this paper is to 1) P1: Provide and discover new insights on bioluminescent bacteria by providing new data and protocols, 2) P2: Genetically modify non-pathogenic bacteria to be bioluminescent to study, 3) P3: Cultivate for further applications and practical uses/innovations. For this study, we are extracting bioluminescent bacteria using an inoculating loop from decayed squid or fish. After resting in ice water for ~3 days, the marine animal will ferment bioluminescent bacterial colonies, in which the light-emitting genome will be extracted. ​ Key Words: Heat shock, bioluminescence, Luciferase, genetic modification, E. Coli K-12 BM032025 - Department of Research 1 RELEASED Introduction ​ Bioluminescent bacteria are a type of microbe that emits light from a series of chemical reactions. The common bacteria species that express bioluminescence are Vibrio and Photobacterium. These species may be stored at 4 degrees Celsius for around 2-4 weeks, preferably submerged in cold water. Light-emitting bacteria are often found in marine habitats, on decayed sea animals, and dinoflagellates. The symbiotic hosts of these bacteria utilize bioluminescence for ecological roles and living functions. For instance, bioluminescent bacteria provide counterillumination camouflage for marine-dwelling organisms, in return for nutrients and shelter. This symbiotic relationship also provides a way for the host to deter predators, attract more hosts for the bacteria, and oxidative stress management. ​ ​ Most bioluminescent bacteria are bacilli and are 1-3 microns long in length. These bacteria strains are often found in seawater environments, populating around 1-100 cells per milliliter of seawater. Bioluminescent bacteria strains are often found in squids, deep-water fish, marine animals, and digestive tracts of fish. This bioluminescent process involves luciferase, aldehyde synthesis, and flavin reductase. The entire lux operon (luxCDABEG) encodes the process for the chain of chemical reactions, in order. Fundamentally, the light is produced from these bacteria through a chemical reaction (FMNH2 + O2 + RCHO → FMN +RCOOH + H2O + Light), where a reduced flavin mononucleotide and molecular oxygen molecule undergoes an enzymatic reaction to form a product of oxidized flavin mononucleotide, water, and light (Brodl et al. par. 18). ​ Luciferin is the substrate in which the Luciferase enzyme catalyzes with. This reaction produces blue-green light. To make a non-pathogenic bacterium bioluminescent, one would need to inject a plasmid (or directly) with the lux operon into a host bacterium. Through heat shock, the pores on the cell membrane of E. Coli would open to allow the DNA in, and close after an ice bath is applied. ​ Using various bioreactor fermentation protocols, E. Coli can be fermented from the 5 milliliter vial into a large half-liter megacolony confined in a glass tank. This biomass will then be harvested and converted into a genetically modified strain of E. Coli that can bioluminesce. For this research study, we are using the E. Coli K-12 strain, an E. Coli strain that is often found in the digestive system. E. Coli K-12 is non-pathogenic, and is frequently used in laboratory studies due to its high genetic tractability and amenability. Because of this, E. Coli K-12 is frequently genetically modified and used as a model eukaryote for studies. ​ Methodology BM032025 - Department of Research 2 RELEASED The methods for treating a bacteria with the lux operon are heat shock, pepsin protein cleaning, ​ The chosen lysis method is via an SDS buffer solution. Section I - Extraction of the Lux Operon and Insertion of the Plasmid into E.Coli K-12 DNA extraction of the Lux Operon requires proteinase, a wash buffer (ethanol/isopropyl-based), magnetic particles for bioconjugation, and spin columns. The method of plasmid insertion was chosen to be Heat Shock via a 42℃ bath, and immediate exposure to an ice bath after 90 seconds. Extraction of the Lux Operon could be performed via genomic lysis; Sodium Dodecyl Sulfate (SDS) and NaOH, or using thermal autolysis. ​ Path I​ ​ The method of extraction is thermal autolysis due to general simplicity and availability of resources, at 55℃. 1 Ml of Proteinase K will be available for the extraction, stored at -20℃ and optimally at 7.5-8.0 pH. Proteinase K will degrade proteins that are attached to the DNA during extraction. ​ Path II - Revised​ Extraction in Path II will be performed via cell lysis using SDS (Sodium Dodecyl Sulfate) and a .5% saline solution. SDS will disrupt the cell membrane to release DNA, while the saline solution will help stabilize the nucleic acids during the extraction process. Section 1A (Optional) - Yeast Fermentation for Protease Extraction To independently produce protease, the yeast Saccharomyces cerevisiae will be fermented for its high protease production. Protease is an enzyme that catalyzes proteolysis, a process to break down the proteins it acts on into individual amino acids. The yeast will be grown and the biomass will be harvested. According to the National Institutes of Health, cell lysis (Thermal Autolysis) will be performed at 47℃ at pH 5.2-6.2. This will lyse the cell through self-digestion activated enzymes. Protease will first be centrifuged and concentrated from beer extract substrates. The substrate cells will then undergo thermal autolysis which will break apart the cell membrane to release the protease. Then, this solution will be centrifuged to separate the precipitate (the protease) from cell wall debris (supernatant). The precipitate will undergo solubilization through sodium hydroxide, and the pH will decrease from 12 to 4.5 so that the solid byproduct of protease is produced. A filter membrane will separate the solid protease from the sodium hydroxide and pH buffer mixture. Section 1B - Lux Operon Extraction BM032025 - Department of Research 3 RELEASED Sterilized with 91% concentrated isopropyl alcohol and left to air dry until dry, fill a centrifuge tube with Vibrio Fischeri inoculated aggregated colonies.A preheated SDS/NaCl solution is added, and the mixture is gently swirled. Reconstituted pepsin is then added, and the sample is incubated at 50–60 °C for 15 minutes to facilitate protein degradation and denaturation, resulting in a purified DNA extract. Chilled 95% ethanol is slowly poured down the side of the tube to form a clear layer above the lysate. White, stringy fibers appear at the interface between the two liquids. Using a sterile stirrer or glass rod, the DNA fibers are gently spooled from the solution and transferred into a tube containing TE buffer for storage. Section II - Bioreactor Fermentation In Bioreactor Fermentation, there is (i) the physical bioreactor itself, and (ii) the fermentation process. The bioreactor fermentor will culture Escherichia Coli K-12 for use in The bioreactor is made of stainless steel, an air filter membrane, silicon tubing, submerged aerator, agitator (rotor), and glass. ​ ​ The bioreactor as well as the inside of the bioreactor will be sterilized beforehand to prevent contamination. We calibrate the probes beforehand to ensure accurate and precise measurements are taken. The fermentation process begins by pouring RPMI 1640 Medium into the bioreactor. The temperature is calibrated to remain at 37℃ / 98.6℉ / 310.15K and the pH will be at 9.25. The bioreactor will have Escherichia Coli K-12 grown in it as the agitator rotates. Section 2A - Impedance-based Cell Counter An external device suspended on the surface of the bioreactor will count the cells in the medium solution (Impedance-based cell counter). The RPMI 1640 Medium contains inorganic salts (sodium, magnesium salts, calcium nitrate, and potassium), allowing electrodes to conduct electricity through the medium and detect resistance (Ω) to count cells. A separate spectrometer or ultrasound device may be used to assume cell density in colonies, but is optional. ​ ​ Some advantages of using an Impedance-based Cell counter is that it calculates particle count with high precision, is energy efficient, and does not require sample staining. However, multiple particles can enter the aperture tube at once, causing a coincidence event and thus skewing the count. Though precision is high, quantitative accuracy is affected due to coincidence and aggregated cell clusters (tissues, combines, etc.). An aperture tube around 10-2000 microns will be suspended within the solution, with the top displaced over the solution. A vacuum will pull cells up into an exterior tube, feeding into the counter driver, to later be pumped back into the medium. The counter driver will cross-analyze impedance and cells to approximate the cell count. One electrode will be put into the aperture tube, and another into the solution. The two electrodes will be connected to a main amplifier, feeding into an oscilloscope and eventually to a counter and digital display. BM032025 - Department of Research 4 RELEASED ​ Section III - Finalized Procedure Step I - Growing Vibrio Fischeri: Submerging squid/fish in an ice bath; 15-28℃. Keep salinity high. Step II - Extracting the Lux operon from Vibrio Fischeri using a DNA Extraction Kit. ​ - Step IIa: Use a SDS (Sodium Dodecyl Sulfate, 7.5%) solution with NaCl (1.5%) to perform cell lysis. - Step IIb: After cell lysis has been performed, employ pepsin to break down proteins. - Step IIc: – N/A, check Instructions Manual when it arrives. (INCOMPLETE) Step III - Centrifuge the lysate to isolate the DNA that is in the spin column. Step IV - Use Ethanol Precipitation to clean any contaminates from the DNA sample. - Step IVa: - ​ Step V - Assess the quality of the cleansed DNA and begin to elute the DNA. Use a Step VI - ​ Materials: ​ 1:https://www.carolina.com/bacteria/escherichia-coli-living-k -12-strain-tube/155068.pr?bvstate=pg:2/ct:r&srsltid=AfmBOor2Y DxlV2okCpqYNfNoaBSaJeX8fy64OYiVGi3Cd5_h6lTOyTb7 ​ 2:https://www.sciencedirect.com/science/article/abs/pii/00928 67483900636 ​ 3:https://amino.bio/products/replacement-bacteria-for-enginee r-it-kit?srsltid=AfmBOoq6VRWHoKaU6DzpMNlx_Xl1tDdlMuHz3OTxB_ze c0HlVEzw2Qzszvs 4: RPMI 1640 Medium https://www.thermofisher.com/order/catalog/product/11875093 5: Lysis Buffer https://www.zymoresearch.com/products/solution-2-lysis-buffer ?srsltid=AfmBOoqJlxlenNSz4T7gF8pQZj894TFn9_DdHV8Jdl8egEf3psy3 cV7lfpw BM032025 - Department of Research 5 RELEASED 6: Proteinase K https://www.elabscience.com/p/proteinase-k-reagent-10-mg-ml-e-ir-r109?srsltid=AfmBOopIfEE7STVSvW7lvrn3pCGJ0HD3O3qQUveqly4 wNdmuDvjTTAW1TpE​ 7: Wash Buffer: https://www.zymoresearch.com/products/m-wash-buffer?srsltid=A fmBOoqZG7DKEyoSy4m-gvRulyptIBQdGmxRxGQMkBeNat5M0fe-lzWmaiY 8: Membrane 0.22 micron filter https://shop.cobetter.com/products/cobetter-nylon-disc-membra ne-filters?variant=44067383542059&utm_source=google&utm_mediu m=cpc&utm_campaign=Google+Shopping&stkn=68be4940826b&srsltid= AfmBOorVr0vdA7Spl1A46TzYOu3RRXn8b-3s84z4ggk0ZRqXmkcXrT2f75U 9: DNA Extraction Kit https://www.amazon.com/DNA-Extraction-Distance-Discovery-Inno vating/dp/B08XWNYV8R/ref=sr_1_4?crid=ME8RM341Y4HQ&dib=eyJ2Ijo iMSJ9.AbnQrq9vJ51TshsmmClTq70vMdN1AvG8YncIkXqMhCdgbcN1zQNQceI GqFL9yixwKAW27k4A0D4P_zKqKHOhhUuKajii3pk4IqqOYyOrtk0pIopOmQDA tGH7h-oIAnSGEOx_-OQ3Fb660vtaQGReQK6a_QNcCE-0hWPildHTeOskR1h0c y4t8e1lq7VzilEJVdOuFGsgTbUYvvrGScQpwNQdfeLbcJHJEdXjhlhTNQs.ir fM_Yb9cvf7BUpXlbYOXVldbc1kXigDew5MYrVOKc0&dib_tag=se&keywords =dna+extraction+kit&qid=1763605595&sprefix=dna+extraction+k%2 Caps%2C469&sr=8-4 ​ Protocol Sources https://education.nationalgeographic.org/resource/bioluminescenc e ​ https://www.the-odin.com/bio-luminescent-bacteria https://prod.cibt.cornell.edu/workshops_and_summer_programs/0608 alum/lumos.pdf ​ Bibliography Brodl, Eveline et al. “Molecular Mechanisms of Bacterial Bioluminescence.” Computational and structural biotechnology journal vol. 16 551-564. 15 Nov. 2018, doi:10.1016/j.csbj.2018.11.003 BM032025 - Department of Research 6 RELEASED Miller, Steven & Haddock, Steven & Elvidge, Christopher & Lee, Thomas. 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Lim, Sun-Joo et al. “Development of Fluorescent Bacteria with Lux and Riboflavin Genes.” International journal of molecular sciences vol. 24,6 5096. 7 Mar. 2023, doi:10.3390/ijms24065096 Mitra, Sagnik, and Ganti S Murthy. “Bioreactor control systems in the biopharmaceutical industry: a critical perspective.” Systems microbiology and biomanufacturing vol. 2,1 (2022): 91-112. doi:10.1007/s43393-021-00048-6 Obom, Kristina M et al. “Operation of a benchtop bioreactor.” Journal of visualized experiments : JoVE ,79 50582. 12 Sep. 2013, doi:10.3791/50582 Jawhara, Samir, and Serge Mordon. “In vivo imaging of bioluminescent Escherichia coli in a cutaneous wound infection model for evaluation of an antibiotic therapy.” Antimicrobial agents and chemotherapy vol. 48,9 (2004): 3436-41. doi:10.1128/AAC.48.9.3436-3441.2004 Marincs, F, and D W White. “Immobilization of Escherichia coli expressing the lux genes of Xenorhabdus luminescens.” Applied and environmental microbiology vol. 60,10 (1994): 3862-3. doi:10.1128/aem.60.10.3862-3863.1994 ​ BM032025 - Department of Research 7 RELEASED Copyright and Intellectual Property Notice: ​ The GPC fully authorizes this document for the following purposes:​ - Constructive criticism - Analysis and constructive feedback - Adding on to research - Essays - Research papers - Education The GPC does not authorize this document for the following purposes: -​ Commercial use -​ Unauthorized distribution​ ​ The GPC does not acquaint itself with partisanism. 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