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Antibiotics

Antibiotics are a class of antimicrobial drugs used to treat and prevent bacterial infections.
They work by inhibiting the growth or killing bacteria, either directly or indirectly.
Antibiotics can be derived from natural sources, such as microorganisms, or synthetically produced.
They are widely used in medicine, agriculture, and animal husbandry to control bacterial diseases.
Antibiotic research and development is an important field, as the rise of antibiotic-resistant bacteria poses a significant threat to public health.
Researchers utilize tools like PubCompare.ai to enhance the reproducibility and accuraacy of their antibiotic studies, leveraging AI-powered comparisons to identify the most effective protocols and products.

Most cited protocols related to «Antibiotics»

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
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
3
Evidence-based Antibiotic Recommendations
The guideline development methodology and how conflict of interest was managed are presented in the online supplement. In brief, the list of PICO questions was finalized based on a prioritization of the most important management decisions balanced against the decision to reduce the overall length of the document and total number of recommendations to maximize readability and usability. We followed the GRADE standards for evaluating the evidence for each PICO and assigned a quality of evidence rating of high, moderate, low, or very low. On the basis of the quality of evidence, recommendations were assigned as strong or conditional. In some cases, strong recommendations were made in the setting of low or very low quality of evidence in accordance with the GRADE rules for when such recommendations are allowable (e.g., when the consequences of the recommendation were high, such as preventing harm or saving life). In all other cases, recommendations that were based on low or very low quality of evidence and not believed to represent standards of care were labeled as conditional recommendations. Statements in favor of strong recommendations begin with the words “We recommend . . .”; statements in favor of conditional recommendations begin with the words “We suggest . . . .” Although we specified pairwise PICO questions for all antibiotic options in the outpatient and inpatient settings, we summarized the recommendations using lists of treatment options, in no preferred order, rather than retain the PICO format for this section.
Metlay J.P., Waterer G.W., Long A.C., Anzueto A., Brozek J., Crothers K., Cooley L.A., Dean N.C., Fine M.J., Flanders S.A., Griffin M.R., Metersky M.L., Musher D.M, & Restrepo M.I. (2019). Diagnosis and Treatment of Adults with Community-acquired Pneumonia. An Official Clinical Practice Guideline of the American Thoracic Society and Infectious Diseases Society of America. American Journal of Respiratory and Critical Care Medicine, 200(7), e45-e67.
Publication 2019
Antibiotics Dietary Supplements Inpatient Outpatients
4
Microdilution Assay for Antimicrobial Activity

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Publication 2007
Antibiotics Asepsis Bacteria Ciprofloxacin Complex Extracts Normal Saline Nutrients resazurin Sterility, Reproductive Strains Sulfoxide, Dimethyl Technique, Dilution
5
Efficient Electroporation and Cryopreservation of Mouse ES Cells
The final targeting constructs were prepared for ES cell electroporation from 2 ml of culture (2X LB plus antibiotics) in 96-well format using the Qiagen Turboprep kit. Before electroporation, vectors were linearized with AsiSI and examined by gel electrophoresis. For most clones, the digested DNA migrated as a single high-molecular-mass band of the expected size (Supplementary Fig. 5). Occasionally, contaminating smaller molecular mass bands were also observed on the gel (DNA quality failures).
JM8 mouse ES cell lines derived from the C57BL/6N strain were grown either on a feeder layer of SNL6/7 fibroblasts (neomycin and/or puromycin resistant) or on gelatinized tissue culture plates16 (link). Both feeder-independent and feeder-dependent lines were maintained in Knockout DMEM (500 ml, Gibco) supplemented with 2 mM glutamine, 5 ml 100× β-mercaptoethanol (360 μl in 500 ml PBS, filter sterilized), 10–15% fetal calf serum respectively (Invitrogen) and 500 U ml−1 leukaemia-inhibitory factor (ESGRO, Millipore). Trypsin solution was prepared by adding 20 ml of 2.5% trypsin solution (Gibco) and 5 ml chicken serum (Gibco) to 500 ml filter-sterilized PBS containing 0.1 g EDTA (Sigma) and 0.5 g d-glucose (Sigma).
Electroporations of ES cells were carried out in a 25-well cuvette using the ECM 630 96-well electroporator /HT-200 automatic plate handler (BTX Harvard Apparatus; set at 700 V, 400 Ω, 25 μF). Immediately before electroporation, cell suspensions of ~1 × 107 cells and ~2 μg of linearized targeting vector DNA were mixed in a final volume of 120 μl PBS. Cells were seeded onto a 10-cm dish (with feeders or gelatin) and colonies were picked after 10 d of selection in 100 μg (active) per ml Geneticin (Invitrogen). To expand cells into duplicate wells for archiving and preparation of genomic DNA, confluent cultures of JM8 ES cells grown on feeder cells were washed twice with pre-warmed PBS and trypsinized for 15 min at 37 °C. Five volumes of pre-warmed media were added and the cells were gently dispersed by tituration and passed at a dilution of 1:4 into new plates containing feeder cells. Passage of cells grown on gelatinized plates was carried out in a similar manner except that the cells were trypsinized for 10 min and passed at a dilution of 1:6 into freshly gelatin-coated plates (0.1% gelatin, Sigma G1393). Culture medium was replaced daily and cells reached confluence 2 days after passage. To archive ES cell clones, trypsinized cells from confluent 96-well plates were transferred in 200 μl freezing medium (Knockout DMEM, 15% serum/ 10% DMSO) to 96-well cryovials (Matrix) and overlayed with sterile mineral oil. The cells were placed at −80 °C overnight and then transferred to liquid nitrogen.
Skarnes W.C., Rosen B., West A.P., Koutsourakis M., Bushell W., Iyer V., Mujica A.O., Thomas M., Harrow J., Cox T., Jackson D., Severin J., Biggs P., Fu J., Nefedov M., de Jong P.J., Stewart A.F, & Bradley A. (2011). A conditional knockout resource for the genome–wide study of mouse gene function. Nature, 474(7351), 337-342.
Publication 2011
2-Mercaptoethanol Antibiotics Cells Chickens Clone Cells Cloning Vectors Edetic Acid Electrophoresis Electroporation Embryonic Stem Cells Feeder Cell Layers Feeder Cells Fetal Bovine Serum Fibroblasts Gelatins Geneticin Genome Glucose Glutamine Hyperostosis, Diffuse Idiopathic Skeletal LIF protein, human Mus Neomycin Nitrogen Oil, Mineral PRSS2 protein, human Puromycin Serum Sterility, Reproductive Strains Sulfoxide, Dimethyl Technique, Dilution Tissues Trypsin

Most recents protocols related to «Antibiotics»

1
Inactivation of Rghr2 Regulated Genes
Not available on PMC !

Example 4

Inactivation of Rghr2 Regulated Genes and Their Effect on Heterologous Protein Production

The Bli03644, abrB1, yvzC and abh genes were inactivated by insertion of antibiotic marker in a Bra7 strain producing a heterologous α-amylase (i.e., the heterologous P. curdlanolyticus α-amylase disclosed in PCT Publication No. WO2014/164834), wherein the heterologous α-amylase production was determined in the four single knock-out strains (ΔBLi03644, ΔabrB1, ΔyvzC and Δabh) and compared to the parental (control) strain as described in Example 2. For example, as presented in FIG. 7, inactivation of Bli03644, abrB1, yvzC and abh resulted in improved heterologous α-amylase production, while cell growth (OD600) was less affected.

US11866713B2. Compositions and methods for increased protein production in bacillus licheniformis (2024-01-09). DANISCO US INC [US]. Inventors: Marc Anton Bernhard Kolkman [NL], Steven D. Doig [US], Ryan L. Frisch [US], Hongxian He [US], Frank Wouter Koopman [NL], Chris Leeflang [NL].
Full text: Click here
Patent 2024
Amylase Antibiotics Bacillus licheniformis Cells Genes Gene Silencing Parent Proteins Strains
2
Chlamydia: Diagnosis, Transmission, and Impacts
Not available on PMC !

Example 2

Chlamydia is a common STI that is caused by the bacterium Chlamydia trachomatis. Transmission occurs during vaginal, anal, or oral sex, but the bacterium can also be passed from an infected mother to her baby during vaginal childbirth. It is estimated that about 1 million individuals in the United States are infected with this bacterium, making chlamydia one of the most common STIs worldwide. Like gonorrhea, chlamydial infection is asymptomatic for a majority of women. If symptoms are present, they include unusual vaginal bleeding or discharge, pain in the abdomen, painful sexual intercourse, fever, painful urination or the urge to urinate more frequently than usual. Of those who develop asymptomatic infection, approximately half may develop PID. Infants born to mothers with chlamydia may suffer from pneumonia and conjunctivitis, which may lead to blindness. They may also be subject to spontaneous abortion or premature birth.

Diagnosis of chlamydial infection is usually done by nucleic acid amplification techniques, such as PCR, using samples collected from cervical swabs or urine specimens (Gaydos et al., J. Clin. Microbio., 42:3041-3045; 2004). Treatment involves various antibiotic regimens.

In some embodiments, the disclosed device can be used to detect chlamydial infections from menstrual blood or cervicovaginal fluids.

US11864740B2. Sample collection and preservation devices, systems and methods (2024-01-09). NextGen Jane, Inc. [US]. Inventors: Ridhi Tariyal [US], Stephen K. Gire [US].
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Patent 2024
Abdominal Pain Antibiotics Anus Asymptomatic Infections Bacteria Blindness Blood Childbirth Chlamydia Chlamydia Infections Chlamydia trachomatis Coitus Conjunctivitis Diagnosis Dysuria Fever Gonorrhea Infant Medical Devices Menstruation Mothers Neck Nucleic Acid Amplification Techniques Pain Patient Discharge Pneumonia Premature Birth Sexually Transmitted Diseases Spontaneous Abortion Transmission, Communicable Disease Treatment Protocols Urine Vagina Woman
3
Combination Therapy Enhances Cancer Cell Killing
Not available on PMC !

Example 25

This experiment was to evaluate the effect of killing cancer cells by treating MDA-MB-231 cells (human breast cancer cells) with the test substance GI-101 alone or in combination with the TGF-beta signal inhibitor Vactosertib substance in an in vitro environment.

MDA-MB-231 cells were purchased from the Korea cell line bank and cultured in RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco). For use in cancer cell killing test, the cells were harvested using trypsin (Gibco), and then suspended in RPMI1640 medium, and then dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution. The cells suspended in RPMI1640 medium were carefully layered on ficoll solution. The cell layer with a low specific gravity formed by centrifuging at room temperature at 350×g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350×g for 5 minutes. The separated cell layer was made into a suspension of 2×105 cells/mL with FBS-free RPMI1640 medium. The cancer cell suspension was stained at 37° C. for 1 hour using CELLTRACKER™ Deep Red Dye (Thermo) in order to track proliferation or inhibition of the proliferation of cancer cells. After staining, it was centrifuged at 1300 rpm for 5 minutes, and then it was washed with FBS-free RPMI1640 medium, and then suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 2×105 cells/mL. The cancer cell suspension was added to each well of a 96-well microplate (Corning) by 50 μl (1×104 cells), and then stabilized in an incubator (37° C., 5% CO2) for 1 hour.

Human peripheral blood mononuclear cells (PBMCs) were used in order to identify the effect of killing cancer cells by GI-101. The human PBMCs were purchased from Zen-Bio, and the PBMCs stored frozen were placed in a 37° C. water bath, and thawed as quickly as possible, and then transferred to RPMI1640 medium (Gibco) containing 10% FBS (Gibco) and 1% antibiotic/antifungal agent (Gibco), and centrifuged at 1300 rpm for 5 minutes. The separated cell layer was suspended in RPMI1640 medium, and then dead cells and debris were removed using Ficoll (GE Healthcare Life Sciences) solution in the same manner as the cancer cell line. The cells suspended in RPMI1640 medium were carefully layered on ficoll solution. The cell layer with a low specific gravity formed by centrifuging at room temperature at 350×g for 20 minutes was collected with a pipette, washed with PBS (Gibco), and then centrifuged at room temperature at 350×g for 5 minutes. The separated cell layer was suspended in RPMI1640 medium containing 5% human AB serum (Sigma) to a concentration of 5×105 cells/mL. The PBMC suspension was dispensed 50 μl into each well of a 96-well microplate (Corning) in which cancer cell line has been dispensed, depending on the conditions.

In order to identify the effect of killing the cells, a CytoTox Green reagent (INCUCYTE™ CytoTox Green, Satorius) that binds to the DNA of cells to be killed was prepared in 1 μl per 1 mL of RPMI1640 medium containing 5% human AB serum (Sigma). The prepared medium was used for dilution of the test substance, and the effect of killing the cells could be quantitatively identified by staining the cells to be killed when the test substance was co-cultured with cancer cell lines and PBMCs.

Vactosertib power was dissolved in DMSO (Sigma) to a concentration of 48.4 mM, and diluted using RPMI1640 medium containing a CytoTox Green reagent, and then used in the experiment at a final concentration of 12.1 nM (50 μL) per well of a 96-well microplate.

GI-101 was diluted by ⅓ using RPMI1640 medium containing a CytoTox Green reagent, and then used in the experiment at final concentrations of 0.4 nM, 1.2 nM, 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM by 50 μl per well of a 96-well microplate.

The prepared test substance was placed in each well of a 96-well microplate in which cancer cell lines and PBMCs were dispensed depending on the conditions, and cultured in an incubator (37° C., 5% CO2) for 24 hours, and the proliferation or death of cancer cells was observed through the real-time cell imaging analysis equipment IncuCyte S3 (Satorious). The death of cancer cells was quantified by the integrated intensity of the cells stained in green with a CytoTox Green reagent.

As a result, it was identified that the group having received a combination of GI-101 and Vactosertib exhibited the excellent effect of killing cancer cells as compared with the group having received each drug alone.

US11857601B2. Pharmaceutical composition for cancer treatment comprising fusion protein including IL-2 protein and CD80 protein and anticancer drug (2024-01-02). GI INNOVATION, INC. [KR]. Inventors: Myung Ho Jang [KR], Su Youn Nam [KR], Young Jun Koh [KR].
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Patent 2024
A-101 Antibiotics Antifungal Agents Bath Cell Death Cell Lines Cell Proliferation Cells Ficoll Freezing Gastrointestinal Cancer Homo sapiens Malignant Neoplasms Mammary Carcinoma, Human MCF-7 Cells MDA-MB-231 Cells PBMC Peripheral Blood Mononuclear Cells PER1 protein, human Pharmaceutical Preparations Psychological Inhibition Serum Sulfoxide, Dimethyl Technique, Dilution Transforming Growth Factor beta Trypsin vactosertib
4
CasX CRISPR Plasmid Interference in E. coli
Not available on PMC !

Example 2

FIGS. 4A-4C. Plasmid Interference by CasX expressed in E. coli. Experimental design of CasX plasmid interference. Competent E. coli cells expressing the minimal interference CasX locus (acquisition proteins removed) were prepared. These cells were transformed with a plasmid containing a match to the spacer in the CasX CRISPR locus (target) or not (non-target) and plated on media containing antibiotic selection for the CRISPR and target plasmid. Successful plasmid interference results in reduced number of transformed colonies for the target plasmid. FIG. 4B cfu/ug of transformed plasmid containing spacer from CasX1 (sX1), spacer from CasX2 (sX2) or a non-target plasmid containing a random 30 nt sequence. FIG. 4C serial dilution was performed of transformants from FIG. 4B on media containing antibiotic selection for both the CRISPR and target plasmid.

FIGS. 5A-5B PAM dependent plasmid interference by CasX. PAM depletion assays were conducted with CasX. E. coli containing the CasX CRISPR locus were transformed with a plasmid library with 7 nucleotides randomized 5′ or 3′ of the target sequence. The target plasmid was selected for and transformants were pooled. The randomized region was amplified and prepared for deep sequencing. Depleted sequences were identified and used to generate a PAM logo. FIG. 5B PAM logo generated for deltaproteobacteria CasX showed a strong preference for sequences containing a 5′-TTCN-3′ flanking sequence 5′ of the target. A 3′ PAM was not detected. c, PAM logo generated for planctomyces CasX showed a strong preference for sequences containing a 5′-TTCN-3′ flanking sequence 5′ of the target with lower stringency at the first T. A 3′ PAM was not detected.

FIGS. 6A-6C. CasX is a dual-guided CRISPR-Cas effector complex. FIG. 6A CRISPR locus for tracrRNA knockout experiments and sgRNA tests. FIG. 6B colony forming units (cfu) per g of transformed plasmid containing a target or non-target sequence. Deletion of the tracrRNA resulted in ablation of plasmid interference. Expression of a synthetic sgRNA in place of the tracrRNA and CRISPR array resulted in robust plasmid interference by CasX. FIG. 6C diagram of sgRNA design (derived from tracrRNA and crRNA sequences for CasX1). The tracrRNA (green) was joined to the crRNA (repeat, black; spacer, red) by a tetraloop (GAAA).

FIG. 7. Schematic of CasX RNA guided DNA interference. CasX binds to a tracrRNA (green) and the crRNA (black, repeat; red, spacer). Base pairing of the guide RNA to the target sequence (blue) containing the correct protospacer adjacent motif (yellow) results in double stranded cleavage of the target DNA. The depicted sequences are derived from tracrRNA and crRNA sequences for CasX1.

US11873504B2. RNA-guided nucleic acid modifying enzymes and methods of use thereof (2024-01-16). The Regents of the University of California [US]. Inventors: Jennifer A Doudna [US], Jillian F. Banfield [US], David Burstein [US], Lucas Benjamin Harrington [US], Steven C. Strutt [US].
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Patent 2024
Antibiotics Biological Assay Cells Clustered Regularly Interspaced Short Palindromic Repeats CRISPR Loci crRNA, Transactivating Deletion Mutation Deltaproteobacteria DNA Cleavage DNA Library Enzymes Escherichia coli Nucleic Acids Nucleotides Plasmids Proteins RNA, CRISPR Guide Technique, Dilution
5
Marker-Free Bacillus subtilis Strain Engineering
Not available on PMC !

Example 1

This example describes the generation of a marker-free B. subtilis strain expressing allulose epimerase. Briefly, in a first step, a B. subtilis strain was transformed with a cassette encoding the BMCGD1 epimerase and including an antibiotic resistance marker. This cassette recombined into the Bacillus chromosome and knocked out 8 kb of DNA, including a large sporulation gene cluster and the lysine biosynthesis gene lysA. In a second step, a second cassette was recombined into the B. subtilis chromosome, restoring the lysA gene and removing DNA encoding the antibiotic resistance. E. coli strain 39 A10 from the Keio collection was used to passage plasmid DNA prior to transformation of B. subtilis. The relevant phenotype is a deficiency in the DNA methylase HsdM in an otherwise wild-type K-12 strain of E. coli.

In detail, a cassette of 5120 bp (SEQ ID NO:1; synthetic DNA from IDT, Coralville, Iowa) was synthesized and cloned into a standard ampicillin resistant pIDT vector. The synthetic piece encoded 700 bp upstream of lysA on the B. subtilis chromosome, the antibiotic marker cat (651 bp), the DNA-binding protein lad (1083 bp), and the allulose epimerase (894 bp), and included 700 bp of homology in dacF. This vector was transformed into E. coli strain 39 A10 (Baba et al., 2006), and plasmid DNA was prepared and transformed into B. subtilis strains 1A751 and 1A976.

Transformants were selected on LB supplemented with chloramphenicol. The replicon for pIDT is functional in E. coli but does not work in Gram positive bacteria such as B. subtilis. The colonies that arose therefore represented an integration event into the chromosome. In strain 1A751, the colony morphology on the plates was used to distinguish between single and double recombination events. The double recombination event would knock out genes required for sporulation, whereas the single recombination would not. After three days on LB plates, colonies capable of sporulation were brown and opaque; sporulation-deficient colonies were more translucent.

B. subtilis strain 1A976 with the allulose epimerase cassette is auxotrophic for histidine and lysine and can achieve very high transformation efficiency upon xylose induction. A 1925 bp synthetic DNA (SEQ ID NO:2) was amplified by primers (SEQ ID NO:3, SEQ ID NO:4) and Taq polymerase (Promega). This PCR product encoded the lysA gene that was deleted by the dropping in the epimerase cassette and 500 bp of homology to lad. A successful double recombination event of this DNA should result in colonies that are prototrophic for lysine and sensitive to chloramphenicol; i.e., the entire cat gene should be lost.

Transformants were selected on Davis minimal media supplemented with histidine. Colonies that arose were characterized by PCR and streaking onto LB with and without chloramphenicol. Strains that amplified the introduced DNA and that were chloramphenicol sensitive were further characterized, and their chromosomal DNA was extracted.

Strain 1A751 containing the chloramphenicol resistant allulose was transformed with this chromosomal DNA and selected on Davis minimal media supplemented with histidine. Transformants were streaked onto LB with and without chloramphenicol and characterized enzymatically as described below.

US11859228B2. Epimerase enzymes and their use (2024-01-02). Archer Daniels Midland Company [US]. Inventors: William Schroeder [US], Padmesh Venkitasubramanian [US], Sridhar Govindarajan [US], Mark Welch [US].
Full text: Click here
Patent 2024
Ampicillin Anabolism Antibiotic Resistance, Microbial Antibiotics Bacillus Bacillus subtilis Chloramphenicol Chromosomes Cloning Vectors DNA, A-Form DNA-Binding Proteins Epimerases Escherichia coli Gene Clusters Gene Knockout Techniques Genes Gram-Positive Bacteria Histidine Lysine Methyltransferase Oligonucleotide Primers Phenotype Plasmids psicose Recombination, Genetic Replicon Strains Taq Polymerase Xylose

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1
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DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium formulated to support the growth and maintenance of a variety of cell types, including mammalian cells. It provides essential nutrients, amino acids, vitamins, and other components necessary for cell proliferation and survival in an in vitro environment.
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Lipofectamine 2000 is a cationic lipid-based transfection reagent designed for efficient and reliable delivery of nucleic acids, such as plasmid DNA and small interfering RNA (siRNA), into a wide range of eukaryotic cell types. It facilitates the formation of complexes between the nucleic acid and the lipid components, which can then be introduced into cells to enable gene expression or gene silencing studies.
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Streptomycin is a broad-spectrum antibiotic used in laboratory settings. It functions as a protein synthesis inhibitor, targeting the 30S subunit of bacterial ribosomes, which plays a crucial role in the translation of genetic information into proteins. Streptomycin is commonly used in microbiological research and applications that require selective inhibition of bacterial growth.
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9
Rpmi 1640 medium by Thermo Fisher Scientific
Sourced in United States, China, Germany, United Kingdom, Japan, France, Canada, Australia, Italy, Switzerland, Belgium, New Zealand, Spain, Israel, Sweden, Denmark, Macao, Brazil, Ireland, India, Austria, Netherlands, Holy See (Vatican City State), Poland, Norway, Cameroon, Hong Kong, Morocco, Singapore, Thailand, Argentina, Taiwan, Province of China, Palestine, State of, Finland, Colombia, United Arab Emirates
RPMI 1640 medium is a commonly used cell culture medium developed at Roswell Park Memorial Institute. It is a balanced salt solution that provides essential nutrients, vitamins, and amino acids to support the growth and maintenance of a variety of cell types in vitro.
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L glutamine by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, France, Canada, Switzerland, Italy, Australia, Belgium, China, Japan, Austria, Spain, Brazil, Israel, Sweden, Ireland, Netherlands, Gabon, Macao, New Zealand, Holy See (Vatican City State), Portugal, Poland, Argentina, Colombia, India, Denmark, Singapore, Panama, Finland, Cameroon
L-glutamine is an amino acid that is commonly used as a dietary supplement and in cell culture media. It serves as a source of nitrogen and supports cellular growth and metabolism.

More about "Antibiotics"

Antibiotics are a class of antimicrobial agents, also known as antibacterial drugs, used to treat and prevent bacterial infections.
These medications work by inhibiting the growth or killing bacteria, either directly or indirectly.
Antibiotics can be derived from natural sources, such as microorganisms, or synthetically produced.
They are widely used in medicine, agriculture, and animal husbandry to control bacterial diseases.
Antibiotic research and development is an important field, as the rise of antibiotic-resistant bacteria poses a significant threat to public health.
Researchers utilize tools like PubCompare.ai to enhance the reproducibility and accuracy of their antibiotic studies, leveraging AI-powered comparisons to identify the most effective protocols and products.
Commonly used in cell culture experiments, antibiotics like Penicillin, Streptomycin, and Antibiotic-antimycotic solutions are often combined with media like FBS, DMEM, RPMI 1640, and L-glutamine to ensure sterile, contamination-free conditions for growing cells.
Lipofectamine 2000 is another important reagent used for transfecting cells with genetic material during research.
Staying up-to-date with the latest advances in antibiotics and leveraging AI-powered tools like PubCompare.ai can help researchers optimize their experimental protocols, improve reproducibility, and make more informed decisions about the most effective antibiotics and antimicrobial agents for their specific research needs.