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Escherichia coli

Escherichia coli: A gram-negative, rod-shaped bacterium that is a common inhabitant of the lower intestine of warm-blooded organisms, including humans.
It is one of the most studdied microorganisms and plays an important role in both medical and biotechnology research.
PubCompare.ai's AI-driven protocol optimization tools can help improve the reproducibility and reliability of E. coli-based experiments, enabling researchers to quickly identify the most effective protocols from scientific literature, preprints, and patents.

Most cited protocols related to «Escherichia coli»

1
Verification of Antimicrobial Resistance Genes
Verification of the databases was made by testing ResFinder with the 1862 GenBank files from which the genes were collected, to verify that the method would find all genes with ID = 100%.
Short sequence reads from 23 isolates of five different species, Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Staphylococcus aureus and Vibrio cholerae, were also submitted to ResFinder. All 23 isolates had been sequenced on the Illumina platform using paired-end reads. A ResFinder threshold of ID = 98.00% was selected, as previous tests of ResFinder had shown that a threshold lower than this gives too much noise (e.g. fragments of genes). Phenotypic antimicrobial susceptibility testing was determined as MIC determinations, as previously described.8 (link)With ‘(chromosome and plasmid)(multi-drug or antimicrobial or antibiotic)(resistant or resistance) pathogen’ as search criteria, one isolate from each species with completely sequenced and assembled, and chromosome and plasmid data were collected from the NCBI Genomes database (http://www.ncbi.nlm.nih.gov/genome). This resulted in 30 isolates, from 30 different species, containing 85 chromosome/plasmid sequences. All sequences were run through all databases in ResFinder with a selected threshold of ID = 98.00%.
Zankari E., Hasman H., Cosentino S., Vestergaard M., Rasmussen S., Lund O., Aarestrup F.M, & Larsen M.V. (2012). Identification of acquired antimicrobial resistance genes. Journal of Antimicrobial Chemotherapy, 67(11), 2640-2644.
Publication 2012
Antibiotics Chromosomes Escherichia coli Genes Genome Klebsiella pneumoniae Microbicides Pathogenicity Pharmaceutical Preparations Phenotype Plasmids Salmonella enterica Staphylococcus aureus Susceptibility, Disease Vibrio cholerae
2
16S rDNA Amplicon and Metagenome Preprocessing
Unassembled sequence reads from both SSU rRNA gene PCR amplicons (pyrotags) and metagenome sequencing were preprocessed (quality control and alignment) by the bioinformatics pipeline of the SILVA project (20 (link)). Briefly, reads shorter than 200 nt or with more than 2% of ambiguities or more than 2% of homopolymers were removed. Remaining reads from amplicons and metagenomes were aligned against the SSU rDNA seed of the SILVA database release 108 (www.arb-silva.de/documentation/background/release-108/) (20 (link)) using SINA (26 (link)). Unaligned reads were not considered in downstream analysis to eliminate non 16S rDNA sequences.
Remaining PCR amplicons were separated based on the presence of aligned nucleotides at E. coli positions of the respective primer binding sites instead of searching for the primer sequences itself. This strategy is robust against sequencing errors within the primer signatures or incomplete primer signatures. This separation strategy works because the amplicon size of one primer pair is significant longer, with overhangs on both 3′ and 5′ site, compared with the amplicon of the second primer pair. With this approach the need for barcoding during combined sequencing of 16S pyrotags derived from different PCR reactions on the same PTP lane was avoided. FASTA files for each primer pair of the separated samples are available online at www.arb-silva.de/download/archive/primer_evaluation.
Reads of the filtered and separated 16S pyrotag datasets as well as metagenomes were dereplicated, clustered and classified on a sample by sample basis. Dereplication (identification of identical reads ignoring overhangs) was done with cd-hit-est of the cd-hit package 3.1.2 (www.bioinformatics.org/cd-hit) using an identity criterion of 1.00 and a wordsize of 8. Remaining sequences were clustered again with cd-hit-est using an identity criterion of 0.98 (wordsize 8). The longest read of each cluster was used as a reference for taxonomic classification, which was done using a local BLAST search against the SILVA SSURef 108 NR dataset (www.arb-silva.de/projects/ssu-ref-nr/) using blast-2.2.22+ (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with default settings. The full SILVA taxonomic path of the best BLAST hit was assigned to the reads if the value for (percentage of sequence identity + percentage of alignment coverage)/2 was at least 93. In the final step, the taxonomic path of each cluster reference read was mapped to the additional reads within the corresponding cluster plus the corresponding replicates (as identified in the previous analysis step) to finally obtain (semi-) quantitative information (number of individual reads representing a taxonomic path). Raw output data are available in the Supplementary Material in Supplementary Tables S48–S50.
Klindworth A., Pruesse E., Schweer T., Peplies J., Quast C., Horn M, & Glöckner F.O. (2012). Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Research, 41(1), e1.
Publication 2012
Binding Sites DNA, Ribosomal Escherichia coli FCER2 protein, human Metagenome Nucleotides Oligonucleotide Primers Ribosomal RNA Genes Sequence Alignment SULT1E1 protein, human
3
Genetic Manipulation of E. coli

E. coli BW25141 (rrnB3 DElacZ4787 DEphoBR580 hsdR514 DE(araBAD)567 DE(rhaBAD)568 galU95 DEendA9::FRT DEuidA3::pir(wt) recA1 rph-1) was used for maintenance of the template plasmid pKD13 (GenBank™ Accession number AY048744). pKD46 (GenBank™ Accession number AY048746; Datsenko and Wanner, 2000 (link)) was made by PCR amplification of the Red recombinase genes from phage λ and cloning into pKD16, a derivative of INT-ts (Haldimann and Wanner, 2001 (link)) carrying araC and araBp from pBAD18 (Guzman et al, 1995 (link)).
Baba T., Ara T., Hasegawa M., Takai Y., Okumura Y., Baba M., Datsenko K.A., Tomita M., Wanner B.L, & Mori H. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology, 2, 2006.0008.
Publication 2006
Ara-C Bacteriophages Escherichia coli Gene Amplification Plasmids Recombinase
4
Evaluating Metagenomic Prediction Accuracy
The accuracy of metagenomic prediction depends on accurate prediction of the gene families (e.g. KOs) present in unsequenced organisms. The accuracy of this gene content prediction step was assessed by using fully sequenced genomes (in which gene content is known) as controls. A test dataset was generated for each sequenced genome in IMG in which that genome was excluded from the reference gene by genome table. PICRUSt was then used to infer the content of the excluded genome. Subsequently, this predicted gene content was compared against the actual gene content, i.e. the sequenced genome annotations. The results were compared using Spearman rank correlation for the actual versus estimated number of gene copies in each gene family or using accuracy and/or balanced accuracy for presence/absence evaluations. These results are presented as the ‘genome holdout’ dataset. In addition to using this dataset to calculate the accuracy of each genome, it was also used to calculate the accuracy per functional gene category. This was done by first mapping KOs to KEGG Modules (described above) for each genome (for both real and PICRUSt predictions) and then comparing each module across all genomes. For visualization, the accuracy of each module was mapped into more general functional categories using the BRITE hierarchy26 (link).
The accuracy of PICRUSt across different taxonomic groups in the phylogenetic tree of bacteria and archaea was visualized using GraPhlAn v0.9 (http://huttenhower.sph.harvard.edu/graphlan). The phylogenetic tree for display was constructed by pruning the Greengenes tree down to tips with corresponding genomes as above, with taxonomic labels at the phylum and genus level obtained for each genome from NCBI Taxonomy49 (link).
We expected that the accuracy of PICRUSt’s predictions would decrease when large phylogenetic distances separated the organism of interest and the nearest sequenced reference genome(s). To test this expectation, ‘distance holdout’ datasets were constructed. These datasets were constructed in the same manner as ‘genome holdout’ datasets described above, except that all genomes within a particular phylogenetic distance (on the 16S tree) of the test organism were excluded from the reference dataset. For example, when predicting Escherichia coli MG1655, a distance holdout of 0.03 substitutions/site would exclude not only that genome, but also all other E. coli strains. These tests were conducted at phylogenetic distances ranging from 0.0 to 0.50 substitutions/site in the full-length 16S rRNA gene, in increments of 0.03 substitutions/site.
Finally, we tested the effects of local inaccuracy in tree construction on PICRUSt’s performance. These ‘tree randomization holdouts’ were constructed the same as the ‘genome holdout’ dataset (above), except that in addition to excluding one genome, the labels of all organisms within a specified phylogenetic distance of the test organism were randomized on the 16S tree. For example, our ‘tree randomization holdout’ targeting E.coli with a distance of 0.03 scrambled the phylogeny of all reference E.coli strains around the tip to be predicted, while leaving the rest of the tree intact. These tests were conducted at phylogenetic distances ranging from 0.0 to 0.50 substitutions/site in the 16S rRNA gene, in increments of 0.03 substitutions/site.
Langille M.G., Zaneveld J., Caporaso J.G., McDonald D., Knights D., Reyes J.A., Clemente J.C., Burkepile D.E., Vega Thurber R.L., Knight R., Beiko R.G, & Huttenhower C. (2013). Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nature biotechnology, 31(9), 814-821.
Publication 2013
Archaea Bacteria Escherichia coli Genes Genome Metagenome Ribosomal RNA Genes Strains Trees
5
Plasmid Construction and Mutagenesis
Plasmid pDESTSIRV30, pDESTSIRV33 expressing the SIRV proteins (CAG38830 and CAG38833), pDESTAVRA expressing MRSA vraR protein (CAG40961) and pDESTFaBH2 expressing Pseudomonas aeruginosa FaBH2 protein (AAG06721)[28 (link)] were constructed using a modified Gateway technology with an N-terminal TEV protease cleavable His tag [29 (link)]. All the plasmids were propagated in DH5α E. coli cells (Stratagene, La Jolla) and plasmids were prepared using Qiagen miniprep kits (Qiagen, Germany). Pfu DNA polymerase, DpnI restriction enzyme are provided with QuikChange™ kit purchased from Stratagene, additional Pfu DNA polymerase was purchased from Promega when required. All the primers were synthesized by Eurogentec and simply purified by SePOP desalting. The melting temperature was calculated as Tm = 81.5 + 16.6(log([K+]/(1+0.7 [K+])) + 0.41(% [G+C]) – 500/(probe length in base) – 1.0(%mismatch) [30 (link)]. The Tm pp and Tm no were calculated for each primer. All primers and their Tm no and Tm pp are detailed in Table 1. PCR cycling was carried out using a Px2 thermal cycler (Thermo Electro Cooperation).
For single-site mutation, deletion or insertion, the PCR reaction of 50 μl contained 2–10 ng of template, 1 μM primer pair, 200 μM dNTPs and 3 units of Pfu DNA polymerase. The PCR cycles were initiated at 95°C for 5 minutes to denature the template DNA, followed by 12 amplification cycles. Each amplification cycle consisted of 95°C for 1 minute, Tm no -5°C for 1 minute and 72°C for 10 minutes or 15 minutes according to the length of the template constructs (about 500 bp per minute for Pfu DNA polymerase). The PCR cycles were finished with an annealing step at Tm pp-5 for 1 minute and an extension step at 72°C for 30 minutes. The PCR products were treated with 5 units of DpnI at 37°C for 2 hours and then 10 μl of each PCR reactions was analyzed by agarose gel electrophoresis. The full-length plasmid DNA was quantified by band density analysis against the 1636-bp band (equal to 10% of the mass applied to the gel) of the DNA ladders. An aliquot of 2 μl above PCR products, the PCR products generated using QuickChange™ or generated as described in [13 (link)] was transformed respectively into E. coli DH5α competent cells by heat shock. The transformed cells were spread on a Luria-Bertani (LB) plate containing antibiotics and incubated at 37°C over night. The number of colonies was counted and used as an indirect indication of PCR amplification efficiency. Four colonies from each plate were grown and the plasmid DNA was isolated. To verify the mutations, 500 ng of plasmid DNA was mixed with 50 pmole of T7 sequencing primer in a volume of 15 μl. DNA sequencing was carried out using the Sequencing Service, University of Dundee. For multiple site-directed mutations, deletions and insertions, the PCR was carried out in 50 μl of reaction containing 10 ng of template, 1 μM of each of the two primer pairs, 200 μM dNTPs and 3 units of Pfu DNA polymerase. The PCR cycles, DNA quantification, transformation and mutation verification were essentially the same as described above.
Liu H, & Naismith J.H. (2008). An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnology, 8, 91.
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Publication 2008
Antibiotics Cells Deletion Mutation DNA Restriction Enzymes Electrophoresis, Agar Gel Escherichia coli Gene Deletion Heat-Shock Response Insertion Mutation Methicillin-Resistant Staphylococcus aureus Mutation Oligonucleotide Primers Pfu DNA polymerase Plasmids Promega Proteins Pseudomonas aeruginosa TEV protease

Most recents protocols related to «Escherichia coli»

1
Translocation of Surface Lipoproteins in E. coli
Not available on PMC !

Example 6

TbpB and NMB0313 genes were amplified from the genome of Neisseria meningitidis serotype B strain B16B6. The LbpB gene was amplified from Neisseria meningitidis serotype B strain MC58. Full length TbpB was inserted into Multiple Cloning Site 2 of pETDuet using restriction free cloning ((F van den Ent, J. Löwe, Journal of Biochemical and Biophysical Methods (Jan. 1, 2006)).). NMB0313 was inserted into pET26, where the native signal peptide was replaced by that of pelB. Mutations and truncations were performed on these vectors using site directed mutagenesis and restriction free cloning, respectively. Pairs of vectors were transformed into E. coli C43 and were grown overnight in LB agar plates supplemented with kanamycin (50 μg/mL) and ampicillin (100 μg/mL).

tbpB genes were amplified from the genomes of M. catarrhalis strain 035E and H. influenzae strain 86-028NP and cloned into the pET52b plasmid by restriction free cloning as above. The corresponding SLAMs (M. catarrhalis SLAM 1, H. influenzae SLAM1) were inserted into pET26b also using restriction free cloning. A 6His-tag was inserted between the pelB and the mature SLAM sequences as above. Vectors were transformed into E. coli C43 as above.

Cells were harvested by centrifugation at 4000 g and were twice washed with 1 mL PBS to remove any remaining growth media. Cells were then incubated with either 0.05-0.1 mg/mL biotinylated human transferrin (Sigma-aldrich T3915-5 MG), α-TbpB (1:200 dilution from rabbit serum for M. catarrhalis and H. influenzae; 1:10000 dilution from rabbit serum for N. meningitidis), or α-LbpB (1:10000 dilution from rabbit serum-obtained a gift from J. Lemieux) or α-fHbp (1:5000 dilution from mouse, a gift from D. Granoff) for 1.5 hours at 4° C., followed by two washes with 1 mL of PBS. The cells were then incubated with R-Phycoerythrin-conjugated Streptavidin (0.5 mg/ml Cedarlane) or R-phycoerythrin conjugated Anti-rabbit IgG (Stock 0.5 mg/ml Rockland) at 25 ug/mL for 1.5 hours at 4° C. The cells were then washed with 1 mL PBS and resuspended in 200 uL fixing solution (PBS+2% formaldehyde) and left for 20 minutes. Finally, cells were washed with 2×1 mL PBS and transferred to 5 mL polystyrene FACS tubes. The PE fluorescence of each sample was measured for PE fluorescence using a Becton Dickinson FACSCalibur. The results were analyzed using FLOWJO software and were presented as mean fluorescence intensity (MFI) for each sample. For N. meningtidis experiments, all samples were compared to wildtype strains by normalizing wildtype fluorescent signals to 100%. Errors bars represent the standard error of the mean (SEM) across three experiments. Results were plotted statistically analysed using GraphPad Prism 5 software. The results shown in FIG. 6 for the SLPs, TbpB (FIG. 6A), LbpB. (FIG. 6B) and fHbp (FIG. 6C) demonstrate that SLAM effects translocation of all three SLP polypeptides in E. coli. The results shown in FIG. 10 demonstrate that translocation of TbpB from M. catarrhalis (FIG. 10C) and in H. influenzae (FIG. 10D) in E. coli require the co-expression of the required SLAM protein (Slam is an outer membrane protein that is required for the surface display of lipidated virulence factors in Neisseria. Hooda Y, Lai C C, Judd A, Buckwalter C M, Shin H E, Gray-Owen S D, Moraes T F. Nat Microbiol. 2016 Feb. 29; 1:16009).

US11872275B2. SLAM polynucleotides and polypeptides and uses thereof (2024-01-16). ENGINEERED ANTIGENS INC. [CA]. Inventors: Trevor F. Moraes [CA], Christine Chieh-Lin Lai [CA], Yogesh Hooda [CA], Andrew Judd [CA], Anthony B. Schryvers [CA], Scott D. Gray-Owen [CA].
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Patent 2024
ADRB2 protein, human Agar Ampicillin anti-IgG Cells Centrifugation Cloning Vectors Culture Media Escherichia coli Fluorescence Formaldehyde Genes Genome Haemophilus influenzae Homo sapiens Kanamycin Lipoproteins Membrane Proteins Moraxella catarrhalis Mus Mutagenesis, Site-Directed Mutation Neisseria Neisseria meningitidis Phycoerythrin Plasmids Polypeptides Polystyrenes prisma Rabbits Serum Signaling Lymphocytic Activation Molecule Family Member 1 Signal Peptides Strains Streptavidin Technique, Dilution Transferrin Translocation, Chromosomal Virulence Factors
2
Identifying SLAM-Dependent Surface Lipoproteins
Not available on PMC !

Example 8

In selecting genomes for a given bacterial species where a SLAM homolog was identified, preference was given to reference genomes that contained fully sequenced genomes. SLAM homologs were identified using iterative Blast searches into closely related species to Neisseria to more distantly related species. For each of the SLAM homologs identified in these species, the corresponding genomic record (NCBI genome) was used to identify genes upstream and downstream along with their corresponding functional annotations (NCBI protein database, Ensembl bacteria). In a few cases, no genes were predicted upstream or downstream of the SLAM gene as they were too close to the beginning or end of the contig, respectively, and thus these sequences were ignored.

Neighbouring genes were analyzed for 1) an N-terminal lipobox motif (predicted using LipoP, SignalP), and 2) a solute binding protein, Tbp-like (InterPro signature: IPR or IPR011250), or pagP-beta barrel (InterPro signature: IPR011250) fold. If they contained these elements, we identified the adjacent genes as potential SLAM-dependent surface lipoproteins.

A putative SLAM (PM1515, SEQ ID NO: 1087) was identified in Pasteurella multocida using the Neisseria SLAM as a search. The putative SLAM (PM1515, SEQ ID NO: 1087) was adjacent to a newly predicted lipoprotein gene with unknown function (PM1514, SEQ ID NO: 1083) (FIG. 11A). The putative SLAM displayed 32% identity to N. meningitidis SLAM1 while the SLP showed no sequence similarity to known SLAM-dependent neisserial SLPs.

The putative SLAM (PM1515, SEQ ID NO: 1087) and its adjacent lipoprotein (PM1514, SEQ ID NO: 1083) were cloned into pET26b and pET52b, respectively, as previously described and transformed into E. coli C43 and grown overnight on LB agar supplemented with kanamycin (50 ug/ml) and ampicillin (100 ug/ml).

Cells were grown in auto-induction media for 18 hours at 37 C and then harvested, washed twice in PBS containing 1 mM MgCl2, and labeled with α-Flag (1:200, Sigma) for 1 hr at 4 C. The cells were then washed twice with PBS containing 1 mM MgCl2 and then labeled with R-PE conjugated α-mouse IgG (25 ug/mL, Thermo Fisher Scientific) for 1 hr at 4 C. following straining, cells were fixed in 2% formaldehyde for 20 minutes and further washed with PBS containing 1 mM MgCl2. Flow Cytometry was performed with a Becton Dickinson FACSCalibur and the results were analyzed using FLOWJO software. Mean fluorescence intensity (MFI) was calculated using at least three replicates was used to compare surface exposure the lipoprotein in strains either containing or lacking the putative SLAM (PM1515) and are shown in FIG. 11C and FIG. 11D. PM1514 could be detected on the surface of E. coli illustrating i) that SLAM can be used to identify SLPs and ii) that SLAM is required to translocate these SLPs to the surface of the cell—thus identifying a class of proteins call “SLAM-dependent surface lipoproteins”. Antibodies were raised against purified PmSLP (PM1514) and the protein was shown to be on the surface of Pasteurella multocida via PK shaving assays.

US11872275B2. SLAM polynucleotides and polypeptides and uses thereof (2024-01-16). ENGINEERED ANTIGENS INC. [CA]. Inventors: Trevor F. Moraes [CA], Christine Chieh-Lin Lai [CA], Yogesh Hooda [CA], Andrew Judd [CA], Anthony B. Schryvers [CA], Scott D. Gray-Owen [CA].
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Patent 2024
Agar Ampicillin Antibodies Bacteria Binding Proteins Biological Assay Cells Escherichia coli Flow Cytometry Fluorescence Formaldehyde Genes Genome Kanamycin Lipoprotein (a-) Lipoproteins Magnesium Chloride Mus Neisseria Neisseria meningitidis Pasteurella multocida Proteins Staphylococcal Protein A Strains
3
Tryptamine's Effect on Gut Motility
Not available on PMC !

Example 6

The organ bath system represents an ex vivo system lacking central nervous system (CNS) connections. Gastrointestinal motility is investigated using mice as an animal model. Experiments are performed to measure colonic contractility in conscious germ free (GF) and colonized mice with infusion of tryptamine by enema as well as following colonization of GF with tryptamine producing E. coli. The effect of tryptamine on epithelial biology also is determined.

US11878002B2. Methods and materials for using Ruminococcus gnavus or Clostridium sporogenes to treat gastrointestinal disorders (2024-01-23). Mayo Foundation for Medical Education and Research [US], The Regents of the University of California [US]. Inventors: Purna C. Kashyap [US], Michael Fischbach [US], Brianna B. Williams [US].
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Patent 2024
Animal Model Bath Central Nervous System Colon Consciousness Enema Escherichia coli Gastrointestinal Motility Mus Muscle Contraction Tryptamines
4
Antibacterial Activity of PABA Analogs
Not available on PMC !

Example 2

Expressed and purified dihydropteroate synthase (DHPS) from S. aureus (saDHPS) was cloned. DHPS is the enzyme that installs PABA (p-aminobenzoic acid) in the folate biosynthesis pathway (Scheme 2). It has been demonstrated that the PABA analog PAS (2-aminosalicylate) is incorporated into folic acid in M. tuberculosis (Chakraborty, S. et al. 2013), suggesting that PAS is a substrate for DHPS. Using a coupled assay, it was determined that the kinetic parameters for saDHPS with PABA, PAS and F-PABA. Importantly, all three compounds have similar kcat and Km values indicating that F-PABA is an alternative substrate for saDHPS. Since PAS is an antibacterial compound whose mechanism of action may be related for the ability of this compound to compete with PABA for DHPS, we determined the antibacterial activity and cytotoxicity of F-PABA for several bacterial species as well as Vero cells. In each case no growth inhibition was observed up to 200 μg/ml. Unlike PAA, 2-F-PABA has no antibacterial activity (Table 1).

[Figure (not displayed)]

TABLE 1
MIC (μg/ml)
2-F-PABAPAS
M. tuberculosis>1000.08
S. aureus>200>200
E. coli>200>200

US11857352B2. Positron imaging tomography imaging agent composition and method for bacterial infection (2024-01-02). The Research Foundation for the State University of New York [US]. Inventors: Peter Tonge [US], Zhuo Zhang [US], Peter Smith-Jones [US], Li Liu [US], Hui Wang [US].
Full text: Click here
Patent 2024
4-Aminobenzoic Acid Anti-Bacterial Agents Bacteria Biological Assay Biosynthetic Pathways Cells Cytotoxin Dihydropteroate Synthase Drug Kinetics Enzymes Escherichia coli Folate Folic Acid Kinetics Mammals Mycobacterium tuberculosis Psychological Inhibition Vero Cells
5
Evolved TpH Background Strain for 5HTP Production
Not available on PMC !

Example 3

Effectiveness of Newly Evolved TpH Background Strain Using Schistosoma mansoni TpH

One of the 7 evolved high 5HTP-producers was selected to further evaluate if the mutations identified were only specifically beneficial to hsTpH2 or could be widely applicable to others. The chosen evolved strain was first cured to lose the evolution plasmid (e.g. the hsTpH gene) and this was immediately followed by re-introducing the E. coli tyrA gene. Upon restoration of the strain's tyrosine auxotrophy, the resulting strain was transformed with pHM2, which is identical to pHM1 used in the earlier evolution study except that the hsTpH gene was replaced with a Schistosoma mansoni TpH gene (SEQ ID NO:9). The 5HTP production of the resulting strain was compared to a wild-type strain carrying pHM2 in the presence of 100 mg/l tryptophan. Results showed the wild-type transformants could only produce ˜0.05 mg/l 5HTP while the newly evolved background strain transformants accumulated >20 mg/l. These production results demonstrated that the mutations acquired in the evolved background strain were not only beneficial to hsTpH but also to other TpHs; possibly applicable also to other aromatic amino acid hydroxylases (e.g. tyrosine hydroxylase).

US11866749B2. Optimized microbial cells for production of melatonin and other compounds (2024-01-09). Danmarks Tekniske Universitet [DK]. Inventors: Hao Luo [DK], Jochen Förster [DK].
Full text: Click here
Patent 2024
5-Hydroxytryptophan Aromatic Amino Acids Biological Evolution Cells Escherichia coli Genes Melatonin Mixed Function Oxygenases Mutation Plasmids Schistosoma mansoni Strains Tryptophan Tyrosine Tyrosine 3-Monooxygenase

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Escherichia coli by American Type Culture Collection
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Escherichia coli is a bacterium that is commonly used in laboratory settings. It serves as a model organism for microbiology and molecular biology research. Escherichia coli can be cultivated and studied to understand fundamental cellular processes and mechanisms.
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More about "Escherichia coli"

Escherichia coli, also known as E. coli, is a gram-negative, rod-shaped bacterium that is a common inhabitant of the lower intestine of warm-blooded organisms, including humans.
It is one of the most extensively studied microorganisms and plays a crucial role in both medical and biotechnology research.
E. coli is a versatile organism that can be leveraged for a wide range of applications, from bioremediation to the production of valuable compounds.
Researchers often utilize E. coli as a model organism to study fundamental biological processes, such as gene expression, protein folding, and cellular metabolism.
One of the key advantages of using E. coli in research is its rapid growth rate and well-characterized genetics.
This makes it an ideal host for cloning and expressing recombinant proteins, such as those derived from the T4 DNA ligase enzyme or the Glutathione Sepharose 4B affinity resin.
To ensure the reliability and reproducibility of E. coli-based experiments, researchers may employ various techniques and tools, including the QIAprep Spin Miniprep Kit for plasmid DNA purification, the PGEM-T Easy vector for cloning, and the Ni-NTA agarose resin for protein purification.
Additionally, the use of antibiotics like ampicillin can help maintain selective pressure and ensure the stability of desired genetic elements within the E. coli cells.
Beyond the laboratory, E. coli is also a significant player in the field of biotechnology.
The bacterium's ability to produce large quantities of proteins, enzymes, and other valuable compounds has made it a workhorse in the production of biofuels, pharmaceuticals, and industrial chemicals.
However, it is important to note that some strains of E. coli, such as those that produce Shiga toxin (referred to as Shiga toxin-producing E. coli or STEC), can pose a serious threat to human health.
Proper safety protocols and containment measures are essential when working with these pathogenic strains.
Overall, the study of E. coli continues to be a crucial area of research, with ongoing advancements in our understanding of its biology and potential applications.
By leveraging the power of PubCompare.ai's AI-driven protocol optimization tools, researchers can streamline their E. coli-based experiments, enhance the reproducibility and reliability of their findings, and contribute to the advancement of this dynamic field of study.