Integrating the extracellular, intracellular, and intercellular pathogenic processes of the microbiome through glucose saturation, inhibition of the acetyl-CoA carboxylase subunit accA with asRNA, and through quantifying cell-to-cell quorum sensing

14 Bacteria ferment the glucose, from fiber, into Short Chain Fatty Acids, which help regulate many biochemical processes and pathways. We cultured Escherichia coli in Luria Broth with 15mM and 5mM concentrations of glucose. The 15mM concentration qPCR measured , for, accA was 4,210 ng/u L. The 7.5u M sample’s concentration equaled 375 ng/u L, and the 0u M sample had an accA concentration of 196 ng/u L. The gene accA, 1 of 4 subunits for the Acetyl-CoA Carboxylase enzyme, was suppressed by asRNA, producing a qPCR concentration of 63ng/u L. Antisense RNA for accA reduced the amount of Lux-S, a vital gene needed for propagating quorum-sensing signal molecules. Our purpose was to provide a more cumulative perspective for pathogenesis of disease within the microbiome.


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The Western diet includes high amounts of fats, sugars, and simple carbohydrates. Due to the Western diet, 16 diabetes, cancer, and many neurological disorders have proliferated and quickly increased the diagnosis 17 of these diseases (1).Ingestion of dietary fiber in the US and in European diets is approximated to be 18 more than a few grams per day. Non-digestible oligosaccharides give between 1 and 2 kcal/g of calories 19 (1). A cause for the prevalence of diseases may be the Western diet that lacks a high source of dietary 20 fiber. There are two main types of dietary fiber, soluble and insoluble fiber. Soluble fiber is found in fruits 21 and vegetables while insoluble fiber includes wheat, cellulose, and inulin. Insoluble fiber is necessary 22 because it maintains a healthy microbiome of the gut by allowing waste in the colon to become bulky 23 for the facile removal of fecal matter from the colon. Insoluble fiber allows for the absorption of water 24 to produce bowel movements more readily without blockage. Fiber regulates and promotes a healthy 25 gut microbiome. A healthy microbiome has many commensal and mutual symbiotic bacterial colonies.

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A well-balanced microbiome prevents colon cancer by the apoptosis of cancerous cells (6). Butyrate, 27 a SCFA, suppresses tumors because it obstructs cell propagation and induces apoptosis when added to 28 different types of tumor cell lines (6). An important bacteria for maintaining homeostasis within the colon 29 and digestive tract, includes E. coli, a gram-negative bacteria that resides within the large intestines (6). E. 30 coli ferments glucose molecules into Butyrate. Butyrate is an attractive therapeutic molecule because 31 of its wide array of biological functions, such as its ability to serve as a histone deacetylase (HDAC) its synthesis. The SCFAs initiate apoptosis(1). Fermentation of glucose to produce SCFAs also constrain 48 the development of disease-causing organisms by decreasing luminal and fecal pH (1). By lessening 49 the pH, the expression of unfavorable bacterial enzymes decreases due to reduced peptide degradation 50 and by the production of ammonia, amines, and phenolic compounds (1). In this study the gene, accA,

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The asRNAs bind to the sequences flanking the ribosome-binding site and the start codon of the target 70 mRNAs (15). They block ribosomes from detecting the RBS, and therefore inhibit translation (15). In 71 the current study, antisense RNA was amplified through PCR of an antisense DNA sequence flanked 72 with Xho1 and Nco1 restriction sites, designing primer sequences of 30bps each. The PCR product 73 was ligated into the PHN1257 plasmid (Fig. 1). Competent bacterial cells were transformed with the 74 recombinant IPTG-PT-asRNA plasmid called PHN1257. The total RNA was extracted for qPCR analysis 75 to determine the number of gene copies for accA. Inhibiting genetic expression, at a intracellular level,

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RNA Preparation Escherichia coli cells were grown on 25mL Luria Broth agar media plates. Transformed 86 bacterial competent cells, with (+antisense)and (-antisense) PTasRNA expression vectors, were grown 87 on LB agar plates with the kanamycin antibiotic and incubated at 37oC for 24hrs. Cells grown on agar 88 plates were inoculated into 4mL of LB liquid media with (+) and (-) PTasRNA bacterial cells expression 89 of asRNA for accA cultured with kanamycin. E.Z.N.A. R Bacterial RNA Kit allowed for rapid and 90 reliable isolation of high-quality total cellular RNA from a wide variety of bacterial species. Up to 3mL 91 of bacterial cell culture from the cells grown in the varied concentrations of glucose, the (+) asRNA, and 92 the (-)asRNA cells were centrifuged at 4,000 x g for 10 minutes at 4oC. The medium was discarded and 93 cells resuspended in 100uL Lysozyme/TE buffer. The solution was vortexed for 30 seconds. Incubaction 94 occured at 30oC for 10 minutes in a shaker-incubator. The lysis buffer of 350uL with 25mg of glass beads 95 were added. It was centrifuged for 5 minutes at maximum speed. RNA was extracted using HiBind R 96 RNA mini columns through RNA wash buffers, I and II. The RNA was eluted with 50uL of DEPC water.  Taq polymerase, we allowed 1 minute for DNA to be amplified at 72 degrees Celsius with a final extension 120 of 5 minutes at 72-74 degrees Celsius. The PCR thermocycler completed 40 cycles of amplification.

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Plasmid Assembly The condition of the PCR products, without primer dimers and contamination, for the 122 target gene accA, were detected through agarose gel electrophoresis. The PCR products were confirmed 123 without primer dimers and contamination, the PCR products and the PTasRNA expression vector of the 124 plasmid PHN1257 were digested with the restriction enzymes XhoI (upstream) and NcoI (downstream) 125 (New England Biolabs XhoI-catalog R0146S-1,000 units and NcoI-catalog R0193S-1,000 units). Each 126 microcentrifuge tube was placed in the incubator for 30 minutes at 37oC. A heat block was heated to 90oC.

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The PCR products of the accA gene were ligated into the PHN1257 plasmid by mixing 1uL of the DNA 128 insert with 2uL of the plasmids, adding 5uL of ligation mix, and then placing the tubes into a heat block

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The RNA Concentration and accA Genetic Expression of each Glucose Sample Escherichia coli MG1655 144 bacteria was grown in LB broth overnight at 37 o C with 15mM, 7.5mM, 5mM, and 0 mM of glucose 145 concentration for high-glucose, medium-glucose, low and zero glucose as a control, respectively (Fig. 2).

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RNA was extracted from each sample and the concentrations determined by the Implen NanoPhotometer 147 250. The RNA concentrations for each sample measured were 1392ng/uL for high-glucose, 797 ng/uL for 148 medium-glucose, 608 ng/u L for low-glucose, and 179 ng/uL for the control. The high-glucose sample 149 had the highest amount of RNA compared to the medium to low and to the control. The RNA for each The qPCR results of asRNA for the target Gene accA Recombinant DNA was produced when the 162 PCR product of the gene insert, accA was ligated into the plasmid PHN1257 ( Fig. 1), that was engineered 163 to amplify antisense RNA. The PCR product and the IPTG-PT-asRNas plasmid of PHN1257 were cut 164 with the restriction enzymes XhoI and NcoI. The primers (Table. 1 to acidic conditions when the luxS/AI-2 system was unregulated. Autoinducer 3 is not dependent on expression of the Lux-S gene. Lux-S genetic mutants impedes the production of AI-3 (31-32). As a result, 303 the pathogenic E.coli, EHEC, collects signals from host cells in the form of hormones as epinephrine.

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AI-3 has the ability to communicate with the transmembrane protein called Qsec (31). Bone marrow 305 white blood cells, as macrophages, can be incubated with synthetic 3-oxo-C12-HSL and C4-HSL, for can decrease genetic expression. In many diseases of the colon, an excess of glucose or hyperglycemia can 357 lead to inhibition of genetic expression of pertinent genes needed for metabolism and fatty acid synthesis. 358 We found that SCFAs, metabolites from the fermentation of insoluble fiber and glucose by E.Coli bacteria, 359 has many beneficial properties and characteristics. Butyrate can bind transmembrane proteins, leading   Table 1. Primers for PCR Amplification and qPCR Analysis. To produce antisense RNA for accA inhibition the restriction enzyme site for XhoI was sequenced with the forward primer for accA. The restriction enzyme NcoI was added to the reverse primer for accA. Through PCR amplification, the pcr products were composed of antisense DNA for the accA gene. The PCR product would be inverted when inserted into the IPTG-PT-asRNA inducible PHN1257 plasmid, forming recombinant DNA plasmids for asRNA transcription in vitro. The plasmids with recombinant antisense DNA were transformed into bacterial competent cells for qPCR analysis of the accA gene. The primers for the Lux-S gene were applied to quantify the number of gene copies from qPCR analysis for cells grown in glucose enhanced samples. The primers for the accA gene were used for the quantification of the accA gene from the samples cultured with an increasing gradient of glucose solutions.

Fig. 2 A)
Glucose concentrations of 15mM, 7.5mM, 5mM, and the control samples each had RNA concentrations, in ng/uL, 1392, 797, 608, 179, respectively. The bacterial sample with 15mM glucose, a high concentration, had the largest measure of RNA, noting a direct proportional link between glucose and genetic expression in gram negative bacteria as E.coli. B) Includes qPCR results for 15mM, 7.5mM, 5mM, and 0mM. The samples with 15mM and 5mM glucose displayed the most genetic activity of accA transcription, measuring accA concentrations at 4,210 ng/uL and 1,500ng/uL respectively. The accA qPCR concentrations for 7.5mM equaled 372 ng/uL and 196 ng/uL for 0mM. C) Displays OD260 results for samples of E.coli grown in medium enhanced with 200uM, 50uM, 0mM of glucose.

Fig. 3 Standard Curves for Quantifying Gene Copies
The RNA for each sample was reverse transcribed into first strand cDNA and absolute quantification with qPCR was used to measure the amount of the target gene, accA, produced by each sample. High-glucose had a Cp of 12.28 and the concentration of accA was 4.21E3 ng/uL. The Cp of sample medium-glucose equaled 16.51 with a concentration of 3.75E2 and the low-glucose Cp was 14.08 with target gene concentration of 1.50E3. The gene of accA was successfully suppressed by asRNA in vitro with 63 ng/uL measured for bacteria cells transformed with the recombinant antisense PHN1257 plasmid DNA. The bacterial cells with the PHN1257 plasmid but without the antisense gene target and insert produced 421.69 ng/uL for accA. There was a 138% percent difference between cells not expressing asRNA versus cells transcribing the asRNA for accA. A p-value of 0.027 showed highly significant data for the accA gene target concentration of PHN1257(+)asRNA versus PHN1257(-)asRNA, or without asRNA.