Operon Construction Containing alsS, ilvC, ilvD and kivd Genes in Esch-erichia coli for Isobutanol Production from Glucose

2020 ◽  
Vol 25 (1) ◽  
pp. 11-17
Author(s):  
Gabriel Kennardi ◽  
◽  
Maelita Moeis ◽  
Andreas Andreas ◽  
◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Acetolactate Synthase ◽  
Gas Chromatography Mass Spectrometry ◽  
Selected Ion Monitoring ◽  
E Coli ◽  
Lac Operator ◽  
Gene Coding ◽  
Sds Page ◽  
Weak Expression ◽  
Lac Promoter

Isobutanol is a biofuel considered to be a potential gasoline substitute. However, isobutanol production is difficult because there is no native organism that can produce isobutanol. A biosynthetic pathway to produce isobutanol had been designed to utilize pyruvate produced from glucose breakdown by glycolysis in Escherichia coli (E. coli). This biosynthetic pathway con-sists of acetolactate-synthase (ALS), ketol-acid reductoisomerase (KARI), dihydroxy-acid dehydratase (DHAD), alpha-ketoisovalerate decarboxylase (KDC) and alcohol dehydrogenase (ADH) enzymes. Since E. coli does not have ALS and KDC, the genes coding for the protein is needed to be cloned and overexpressed in E. coli. KARI and DHAD were overexpressed to increase the accumulation of keto acid to increase isobutanol production. Plasmid contains an operon controlled by lac pro-moter and lac operator consisting of alsS (coded ALS from Bacillus subtilis), ilvC (coded KARI from E. coli MG1655) and ilvD (coded DHAD from E. coli MG1655) genes, obtained from previous research, and operon sequences have been confirmed by DNA sequencing. kivd gene (coding KDC from Lactococcus lactis) was obtained from iGEM 2013 kit. kivd was amplified by PCR and inserted into pJET 1.2 blunt. kivd gene was then added into 3’ end of previous operon using restriction-ligation tech-nique. The plasmid constructed was then transfered into E. coli DH5α using heat shock. The recombinant genes were expressed using IPTG (isopropyl-β-D-1-thiogalactopyranoside) induction. The SDS PAGE results were inconclusive, however isobutanol was detected by Gas Chromatography Mass Spectrometry – Selected Ion Monitoring (GC-MS-SIM) from 48 hours fermenta-tion culture at 30 oC (1,17%). An operon regulated by the lac promoter-operator containing four genes for the biosynthesis of isobutanol has been constructed and cloned in E. coli. The isobutanol production was not optimal due to weak expression and repression by glucose, which was used as substrate.

scholarly journals VERIFIKASI cDNA T29 SEBAGAI KANDIDAT GEN PENGKODE PROTEIN Toxoplasma gondii DENGAN METODE SDS PAGE

2005 ◽  
Vol 11 (1) ◽  
pp. 61-66
Author(s):  
Ira Djajanegara ◽  
Wayan Artama ◽  
Retno Lestari ◽  
Sabar Pambudi
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Plasmid ◽  
Restriction Site ◽  
Standard Procedure ◽  
Pcr Amplification ◽  
Gene Encoding ◽  
E Coli ◽  
Gene Coding ◽  
Sds Page ◽  
Encoding Protein

The process of cDNA construction from mRNA isolated from Toxoplasma gondii has been done. There were 7 candidates cDNA which one of them is called T29. Since Toxoplasma gondii is the cause of toxoplasmosis infection, cloning the gene encoding protein from this parasite provides an important tool for developing diagnostic kit for detection of toxoplasmosis. Digestion of the cDNA T29 with EcoRI which is the restriction site where the cDNA was inserted yielded a 1.862 bp fragment. The fragment was subcloned into E. coli expression vector pMal-p2x and transformed into E.coli strain TB1. Colonies of TB1 were grown on ampicillin plates and the recombinant plasmid was extracted using the standard procedure. The plasmid was digested using EcoRI and PstI, checked by PCR amplification using malE and M13/pUC primers. The recombinant plasmid was expressed in TB1 and the protein extracted was ran in SDS PAGE to observe the presence of the expressed protein. Based on the data from this experiment, there was no expression result of the expressed cDNA which was confirm by the PCR result. Therefore, it was concluded that cDNA T29 was not carrying the gene coding for protein from parasite Toxoplasma gondii.

scholarly journals Expression in Escherichia coli and characterization of a recombinant 7Fe ferredoxin of Rhodobacter capsulatus

1992 ◽  
Vol 286 (1) ◽  
pp. 269-273 ◽  
Author(s):  
Y Jouanneau ◽  
C Duport ◽  
C Meyer ◽  
J Gaillard
Keyword(s):  
Escherichia Coli ◽  
Rhodobacter Capsulatus ◽  
Spectroscopic Properties ◽  
Coli Cell ◽  
E Coli ◽  
Expression In Escherichia Coli ◽  
Gene Coding ◽  
Lac Promoter

The 7Fe ferredoxin of Rhodobacter capsulatus (FdII) could be expressed in Escherichia coli by cloning the fdxA gene coding for FdII downstream from the lac promoter. The expressed recombinant ferredoxin appeared as a brown protein which was specifically recognized in E. coli cell-free extracts by anti-FdII serum. The purified recombinant ferredoxin was indistinguishable from R. capsulatus FdII on the basis of its molecular, redox and spectroscopic properties. These results indicate that the [3Fe-4S] and [4Fe-4S] clusters were correctly inserted into the recombinant ferredoxin.

Enhanced Biocatalytic Activity of Recombinant Lipase Immobilized on Gold Nanoparticles

2019 ◽  
Vol 20 (6) ◽  
pp. 497-505 ◽  
Author(s):  
Abeer M. Abd El-Aziz ◽  
Mohamed A. Shaker ◽  
Mona I. Shaaban
Keyword(s):  
Gold Nanoparticles ◽  
Lipolytic Activity ◽  
Market Demand ◽  
Biotechnological Applications ◽  
Lipase Gene ◽  
Expression Plasmid ◽  
E Coli ◽  
Nickel Affinity Chromatography ◽  
Recombinant Production ◽  
Sds Page

Background: Bacterial lipases especially Pseudomonas lipases are extensively used for different biotechnological applications. Objectives: With the better understanding and progressive needs for improving its activity in accordance with the growing market demand, we aimed in this study to improve the recombinant production and biocatalytic activity of lipases via surface conjugation on gold nanoparticles. Methods: The full length coding sequences of lipase gene (lipA), lipase specific foldase gene (lipf) and dual cassette (lipAf) gene were amplified from the genomic DNA of Pseudomonas aeruginosa PA14 and cloned into the bacterial expression vector pRSET-B. Recombinant lipases were expressed in E. coli BL-21 (DE3) pLysS then purified using nickel affinity chromatography and the protein identity was confirmed using SDS-PAGE and Western blot analysis. The purified recombinant lipases were immobilized through surface conjugation with gold nanoparticles and enzymatic activity was colorimetrically quantified. Results: Here, two single expression plasmid systems pRSET-B-lipA and pRSET-B-lipf and one dual cassette expression plasmid system pRSET-B-lipAf were successfully constructed. The lipolytic activities of recombinant lipases LipA, Lipf and LipAf were 4870, 426 and 6740 IUmg-1, respectively. However, upon immobilization of these recombinant lipases on prepared gold nanoparticles (GNPs), the activities were 7417, 822 and 13035 IUmg-1, for LipA-GNPs, Lipf-GNPs and LipAf-GNPs, respectively. The activities after immobilization have been increased 1.52 and 1.93 -fold for LipA and LipAf, respectively. Conclusion: The lipolytic activity of recombinant lipases in the bioconjugate was significantly increased relative to the free recombinant enzyme where immobilization had made the enzyme attain its optimum performance.

Comparison of the Effects of Silver in Nanostructured and Ultrahigh Diluted Form on Growth and Volatile Compounds Produced by Escherichia coli and Staphylococcus aureus

2020 ◽  
Vol 10 (3) ◽  
pp. 316-329
Author(s):  
Fateme Mirzajani ◽  
Amin Hamidi
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Life Cycles ◽  
Gas Chromatography Mass Spectrometry ◽  
Untreated Sample ◽  
E Coli ◽  
Volatile Metabolites ◽  
Octyl Acetate ◽  
Volatile Organic ◽  
And Staphylococcus Aureus

Introduction: In this project, the growth and volatile metabolites profiles of Escherichia coli (E. coli ) and Staphylococcus aureus were monitored under the influence of silver base chemical, nanoparticle and ultra-highly diluted compounds. Materials & Methods: The treatments were done for 12000 life cycles using silver nanoparticles (AgNPs) as well as ultra-highly diluted Argentum nitricum (Arg-n). Volatile organic metabolites analysis was performed using gas chromatography mass spectrometry (GC-MS). The results indicated that AgNPs treatment made the bacteria resistant and adapted to growth in the nanoparticle condition. The use of ultra-highly diluted Arg-n initially increased growth but it decreased later. Also, with the continuous usage of these materials, no more bacterial growth was observed. Results: The most important compounds produced by E. coli are Acetophenone, Octyl acetate, Styrene, 1,8-cineole, 4-t-butyl-2-(1-methyl-2-nitroethyl)cyclohexane, hexadecane and 2-Undecanol. The main compounds derived from S. aureus are Acetophenone,1,8-cineole, Benzaldehyde, 2-Hexan-1-ol, Tridecanol, Dimethyl Octenal and tetradecane. Acetophenone and 1,8-cineole were common and produced by both organisms. Conclusion: Based on the origin of the produced volatiles, main volatiles percentage of untreated sample is hydrocarbon (>50%), while bacteria treatments convert the ratio in to aldehydes, ketones and alcohols in the case of AgNPs, (>80%) and aldehydes, ketones and terpenes in the case of Arg-n (>70%).

Characterization of drug interferences caused by coelution of substances in gas chromatography/mass spectrometry confirmation of targeted drugs in full-scan and selected-ion monitoring modes

1994 ◽  
Vol 40 (2) ◽  
pp. 216-220 ◽  
Author(s):  
A H Wu ◽  
D Ostheimer ◽  
M Cremese ◽  
E Forte ◽  
D Hill
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography Mass Spectrometry ◽  
Spectrometric Analysis ◽  
Targeted Drugs ◽  
Selected Ion Monitoring ◽  
Internal Standards ◽  
Target Drug ◽  
Full Scan ◽  
Chromatographic Mass

Abstract Interference by substances coeluting with targeted drugs is a general problem for gas chromatographic/mass spectrometric analysis of urine. To characterize these interferences, we examined human urine samples containing benzoylecgonine and fluconazole, and other drug combinations including deuterated internal standards that coelute (ISd,c) with target drugs, by selected-ion monitoring (SIM) and full-scan mass spectrometry. We show that, by SIM analysis, detecting the presence of an interferent is dependent on the specific IS used for the assay. When an ISd,c is used, the presence of another coeluting substance (interferent) suggests that the intensity of IS ions is substantially diminished, because the interferent affects both the ISd,c and target drug. When a noncoeluting IS (ISnc) is used, the interferent cannot be discerned unless it coincidently contains one or more of the ions monitored for either the target drug or ISnc. Under full-scan analysis, a coeluting interferent is directly discernable by examining the total ion gas chromatogram.

scholarly journals A NEW MAP LOCATION FOR THE ilvB LOCUS OF ESCHERICHIA COLI

Genetics ◽  
1980 ◽  
Vol 96 (1) ◽  
pp. 59-77
Author(s):  
Thomas C Newman ◽  
Mark Levinthal
Keyword(s):  
Escherichia Coli ◽  
Structural Gene ◽  
Acetolactate Synthase ◽  
Deletion Mapping ◽  
Donor Strain ◽  
E Coli ◽  
Linkage Of Genes ◽  
Map Location ◽  
New Alleles

ABSTRACT We isolated, in E. coli K12, new alleles of the ilvB locus, the structural gene for acetolactate synthase isoenzyme I, and showed them to map at or near the ilvB619 site. The map position of the ilvB locus was redetermined because plasmids containing the ilvC-cya portion of the chromosome did not complement mutations at the ilvB locus. Furthermore, diploids for the ilvEDAC genes formed with these plasmids in an ilvHI background facilitated the isolation of the new ilvB alleles. The ilvB locus was remapped and found to be located at 81.5 minutes, between the uhp and dnaA loci. This location was determined by two- and three-point transductional crosses, deletion mapping and complementation with newly isolated plasmids. One of the new alleles of the ilvB gene is a mu-1 insertion. When present in the donor strain, this allele interferes with the linkage of genes flanking the mu-1 insertion, as well as the linkage of genes to either side of the mu-1 insertion.

scholarly journals Transcriptome and Metabolome Dynamics Explain Aroma Differences between Green and Red Prickly Ash Fruit

Foods ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 391
Author(s):  
Xitong Fei ◽  
Yichen Qi ◽  
Yu Lei ◽  
Shujie Wang ◽  
Haichao Hu ◽  
...  
Keyword(s):  
Redundancy Analysis ◽  
Biosynthetic Pathway ◽  
Expression Patterns ◽  
Gas Chromatography Mass Spectrometry ◽  
Zanthoxylum Armatum ◽  
Aroma Characteristics ◽  
Main Factors ◽  
Key Genes ◽  
Zanthoxylum Bungeanum ◽  
Aroma Components

Green prickly ash (Zanthoxylum armatum) and red prickly ash (Zanthoxylum bungeanum) fruit have unique flavor and aroma characteristics that affect consumers’ purchasing preferences. However, differences in aroma components and relevant biosynthesis genes have not been systematically investigated in green and red prickly ash. Here, through the analysis of differentially expressed genes (DEGs), differentially abundant metabolites, and terpenoid biosynthetic pathways, we characterize the different aroma components of green and red prickly ash fruits and identify key genes in the terpenoid biosynthetic pathway. Gas chromatography-mass spectrometry (GC-MS) was used to identify 41 terpenoids from green prickly ash and 61 terpenoids from red prickly ash. Piperitone was the most abundant terpenoid in green prickly ash fruit, whereas limonene was most abundant in red prickly ash. Intergroup correlation analysis and redundancy analysis showed that HDS2, MVK2, and MVD are key genes for terpenoid synthesis in green prickly ash, whereas FDPS2 and FDPS3 play an important role in the terpenoid synthesis of red prickly ash. In summary, differences in the composition and content of terpenoids are the main factors that cause differences in the aromas of green and red prickly ash, and these differences reflect contrasting expression patterns of terpenoid synthesis genes.

scholarly journals Application of Bar Adsorptive Microextraction for the Determination of Levels of Tricyclic Antidepressants in Urine Samples

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3101
Author(s):  
Mariana N. Oliveira ◽  
Oriana C. Gonçalves ◽  
Samir M. Ahmad ◽  
Jaderson K. Schneider ◽  
Laiza C. Krause ◽  
...  
Keyword(s):  
Tricyclic Antidepressants ◽  
Matrix Effects ◽  
Activated Carbons ◽  
Process Efficiency ◽  
Gas Chromatography Mass Spectrometry ◽  
Selected Ion Monitoring ◽  
Experimental Conditions ◽  
Analytical Methodology ◽  
Low Efficiency

This work entailed the development, optimization, validation, and application of a novel analytical approach, using the bar adsorptive microextraction technique (BAμE), for the determination of the six most common tricyclic antidepressants (TCAs; amitriptyline, mianserin, trimipramine, imipramine, mirtazapine and dosulepin) in urine matrices. To achieve this goal, we employed, for the first time, new generation microextraction devices coated with convenient sorbent phases, polymers and novel activated carbons prepared from biomaterial waste, in combination with large-volume-injection gas chromatography-mass spectrometry operating in selected-ion monitoring mode (LVI-GC-MS(SIM)). Preliminary assays on sorbent coatings, showed that the polymeric phases present a much more effective performance, as the tested biosorbents exhibited low efficiency for application in microextraction techniques. By using BAμE coated with C18 polymer, under optimized experimental conditions, the detection limits achieved for the six TCAs ranged from 0.2 to 1.6 μg L−1 and, weighted linear regressions resulted in remarkable linearity (r2 > 0.9960) between 10.0 and 1000.0 μg L−1. The developed analytical methodology (BAμE(C18)/LVI-GC-MS(SIM)) provided suitable matrix effects (90.2–112.9%, RSD ≤ 13.9%), high recovery yields (92.3–111.5%, RSD ≤ 12.3%) and a remarkable overall process efficiency (ranging from 84.9% to 124.3%, RSD ≤ 13.9%). The developed and validated methodology was successfully applied for screening the six TCAs in real urine matrices. The proposed analytical methodology proved to be an eco-user-friendly approach to monitor trace levels of TCAs in complex urine matrices and an outstanding analytical alternative in comparison with other microextraction-based techniques.

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