scholarly journals An exploration of the rapid transformation method for Dunaliella salina system

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Guannan Song ◽  
Wan Wang ◽  
Lina Hu ◽  
Yu Liu ◽  
Aifang Li ◽  
...  
Keyword(s):  
Dunaliella Salina ◽  
Genetic Manipulation ◽  
Expression System ◽  
Transformation Method ◽  
Pcr Analysis ◽  
Foreign Gene ◽  
High Efficient ◽  
Set Up ◽  
Transformation Methods ◽  
Rapid Transformation

Abstract As a new expression system, Dunaliella salina (D. salina) has bright prospects and applications in various fields. However, its application is currently restricted because of the low expression and instability of foreign gene in D. salina cells. During genetic operation, transformation is a crucial step for genes expression in D. salina system. Although several transformation methods are existing currently, many inherent deficiencies and limitations still can be found in actual practice. Thus, we attempted to set up a rapid transformation method using the change of salt concentrations for D. salina. Based on osmotic pressure difference, exogenous genes can be spontaneously transferred into D. salina cells. After that, transformed D. salina cells were subjected to histochemical and molecular analysis. The results showed that the reporter gene, beta-glucuronidase genes were successfully expressed in the positive transformants, and detected in all of transformed cells by PCR analysis. Moreover, different transformation parameters, containing the salt gradient, time, dye dosage and Triton X-100 concentration, were optimized to obtain an optimal transformation result. Taken together, we preliminarily established a rapid transformation method with the features of fast, simple, economic, and high-efficient. This method will provide a strong genetic manipulation tool for the future transformation of D. salina system.

CRISPR/Cas9-induced β-carotene Hydroxylase Mutation in Dunaliella Salina CCAP19/18

2021 ◽  
Author(s):  
Lina Hu ◽  
Shu ying FENG ◽  
Gaofeng Liang ◽  
Jingxia Du ◽  
Aifang Li ◽  
...  
Keyword(s):  
High Performance ◽  
Dunaliella Salina ◽  
Gene Editing ◽  
Expression System ◽  
Transformation Method ◽  
Practical Significance ◽  
Exon 1 ◽  
Targeted Gene Editing ◽  
Foreign Genes ◽  
Β Carotene

Abstract Dunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of CRISPR/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina. This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina, and then gives a strong tool to facilitates the development and application of D. salina system.

scholarly journals CRISPR/Cas9-induced β-carotene hydroxylase mutation in Dunaliella salina CCAP19/18

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lina Hu ◽  
Shuying Feng ◽  
Gaofeng Liang ◽  
Jingxia Du ◽  
Aifang Li ◽  
...  
Keyword(s):  
High Performance ◽  
Dunaliella Salina ◽  
Gene Editing ◽  
Expression System ◽  
Transformation Method ◽  
Practical Significance ◽  
Exon 1 ◽  
Short Palindromic Repeat ◽  
Foreign Genes ◽  
Β Carotene

AbstractDunaliella salina (D. salina) has been exploited as a novel expression system for the field of genetic engineering. However, owing to the low or inconsistent expression of target proteins, it has been greatly restricted to practical production of recombinant proteins. Since the accurate gene editing function of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system, β-carotene hydroxylase gene was chosen as an example to explore D. salina application with the purpose of improving expression level of foreign genes. In this paper, based on pKSE401 backbone, three CRISPR/Cas9 binary vectors were constructed to targeting exon 1 and 3 of the β-carotene hydroxylase of D. salina CCAP19/18 (Dschyb). D. salina mutants were obtained by salt gradient transformation method, and the expression of Dschyb gene were identified through real-time fluorescent quantitative PCR. Moreover, carotenoids content was analyzed by high-performance liquid chromatography at different time points after high intensity treatment. Compared with wild type strains, the β-carotene levels of mutants showed a significant increase, nearly up to 1.4 μg/ml, and the levels of zeaxanthin decreased to various degrees in mutants. All the results provide a compelling evidence for targeted gene editing in D. salina. This study gave a first successful gene editing of D. salina which has a very important practical significance for increasing carotene yield and meeting realistic industry demand. Furthermore, it provides an approach to overcome the current obstacles of D. salina, and then gives a strong tool to facilitates the development and application of D. salina system.

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

scholarly journals A new method for identifying the role of marital preferences at shaping marriage patterns

2021 ◽  
pp. 1-27
Author(s):  
Anna Naszodi ◽  
Francisco Mendonca
Keyword(s):  
Contingency Table ◽  
Transformation Method ◽  
New Method ◽  
Marriage Patterns ◽  
The Us ◽  
Analytical Properties ◽  
The Matrix ◽  
Transformation Methods

Abstract We develop a method which assumes that marital preferences are characterized either by the scalar-valued measure proposed by Liu and Lu, or by the matrix-valued generalized Liu–Lu measure. The new method transforms an observed contingency table into a counterfactual table while preserving its (generalized) Liu–Lu value. After exploring some analytical properties of the new method, we illustrate its application by decomposing changes in the prevalence of homogamy in the US between 1980 and 2010. We perform this decomposition with two alternative transformation methods as well where both methods capture preferences differently from Liu and Lu. Finally, we use survey evidence to support our claim that out of the three considered methods, the new transformation method is the most suitable for identifying the role of marital preferences at shaping marriage patterns. These data are also in favor of measuring assortativity in preferences à la Liu and Lu.

scholarly journals Random numbers from the tails of probability distributions using the transformation method

2013 ◽  
Vol 16 (2) ◽  
Author(s):  
Daniel Fulger ◽  
Enrico Scalas ◽  
Guido Germano
Keyword(s):  
Probability Distributions ◽  
Transformation Method ◽  
Fractional Diffusion ◽  
Random Numbers ◽  
Stochastic Solution ◽  
Probability Densities ◽  
Speed Up ◽  
Time Fractional Diffusion Equation ◽  
Transformation Methods ◽  
Very High

AbstractThe speed of many one-line transformation methods for the production of, for example, Lévy alpha-stable random numbers, which generalize Gaussian ones, and Mittag-Leffler random numbers, which generalize exponential ones, is very high and satisfactory for most purposes. However, fast rejection techniques like the ziggurat by Marsaglia and Tsang promise a significant speed-up for the class of decreasing probability densities, if it is possible to complement them with a method that samples the tails of the infinite support. This requires the fast generation of random numbers greater or smaller than a certain value. We present a method to achieve this, and also to generate random numbers within any arbitrary interval. We demonstrate the method showing the properties of the transformation maps of the above mentioned distributions as examples of stable and geometric stable random numbers used for the stochastic solution of the space-time fractional diffusion equation.

Production of foreign proteins in the yeast Pichia pastoris

1995 ◽  
Vol 73 (S1) ◽  
pp. 891-897 ◽  
Author(s):  
James M. Cregg ◽  
David R. Higgins
Keyword(s):  
Gene Expression ◽  
Pichia Pastoris ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Methylotrophic Yeast ◽  
Culture Broth ◽  
Heterologous Gene ◽  
Foreign Gene ◽  
Heterologous Proteins ◽  
Yeast Pichia Pastoris

The methanol-utilizing yeast Pichia pastoris has been developed as a host system for the production of heterologous proteins of commercial interest. An industrial yeast selected for efficient growth on methanol for biomass generation, P. pastoris is readily grown on defined medium in continuous culture at high volume and density. A unique feature of the expression system is the promoter employed to drive heterologous gene expression, which is derived from the methanol-regulated alcohol oxidase I gene (AOX1) of P. pastoris, one of the most efficient and tightly regulated promoters known. The strength of the AOX1 promoter results in high expression levels in strains harboring only a single integrated copy of a foreign-gene expression cassette. Levels may often be further enhanced through the integration of multiple cassette copies into the P. pastoris genome and strategies to construct and select multicopy cassette strains have been devised. The system is particularly attractive for the secretion of foreign-gene products. Because P. pastoris endogenous protein secretion levels are low, foreign secreted proteins often appear to be virtually the only proteins in the culture broth, a major advantage in processing and purification. Key words: heterologous gene expression, methylotrophic yeast, Pichia pastoris, secretion, glycosylation.

scholarly journals 1724. Plasmid-free CRISPR-Cas9 System for Genetic Engineering of Rhizopus delemar

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S632-S632
Author(s):  
Yiyou Gu ◽  
Clara Baldin ◽  
Teklegiorgis Gebremariam ◽  
Abdullah Alqarihi ◽  
Zeinab Mamouei ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Etiologic Agent ◽  
Pcr Analysis ◽  
Free System ◽  
Rhizopus Delemar ◽  
Additional Tool ◽  
Qrt Pcr ◽  
Enzyme Screening ◽  
Serious Infection ◽  
Short Palindromic Repeat

Abstract Background Mucormycosis is a serious infection caused by fungi of the order Mucorales. Rhizopus delemar is the most common etiologic agent of mucormycosis. Pathogenesis studies of mucormycosis have been hampered by poor genetic trackability of the organism, owing to rare chromosomal integration events and multinucleated nature of the cells. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system has been widely used in genetic manipulation through efficient homologous and non-homologous break points in a variety of organisms including R. delemar. However, plasmid-free CRISPR/Cas9 system has not been previously described in the fungus. Here, we introduce a rapid plasmid-free system for inducing orotidine 5’-phosphate decarboxylase (pyrF) gene mutation in R. delemar. Methods Protoplasts of R. delemar 99–880 strain were transformed with 20 nucleotide gRNA targeting the N-terminus of pyrF gene and the Cas9 enzyme. Screening for pyrF auxotrophy was carried out by plating transformed protoplasts on potato dextrose agar (PDA) plates containing 1 mg/mL 5-fluoroorotic acid (5-FOA) and 100 µg/mL uracil. Putative mutant strains were selected for uracil auxotrophy by plating simultaneously on media with or without uracil. pyrF disruption was verified by using PCR and qRT–PCR. Results Approximately100 transformants were generated through plating on 5-FOA plates. Only three transformants did not grow on minimal medium lacking uracil, indicating that they were true pyrF null mutants. PCR analysis showed that these three transformants have undergone nucleotide deletion events within the pyrF gene. The lack of pyrF gene expression was further verified by using qRT–PCR relative to wild-type R. delemar 99–880. Conclusion Similar to the plasmid-based genome manipulation strategy, the plasmid-free CRISPR/Cas9 system can induce gene editing in R. delemar. This rapid and simple approach adds an additional tool in our conquest to understand pathogenesis of mucormycosis. Disclosures All authors: No reported disclosures.

Production of Lysinibacillus sphaericus Mosquitocidal Protein Mtx2 from Bacillus subtilis as a Secretory Protein

2021 ◽  
Vol 28 ◽  
Author(s):  
Chutchanun Trakulnaleamsai ◽  
Boonhiang Promdonkoy ◽  
Sumarin Soonsanga
Keyword(s):  
Bacillus Subtilis ◽  
Larvicidal Activity ◽  
Mosquito Control ◽  
Expression System ◽  
Secretory Protein ◽  
Signal Peptides ◽  
Secreted Protein ◽  
Lysinibacillus Sphaericus ◽  
Putative Signal Peptide ◽  
Set Up

Background: Mtx2 is a mosquitocidal toxin produced during the vegetative growth of Lysinibacillus sphaericus. The protein shows synergism with other toxins against mosquito larvae; hence it could be used in mosquito control formulations. The protein expression system is needed for Mtx2 development as a biocontrol agent. Objective: The objective of the study was to set up a Bacillus subtilis system to produce Mtx2 as a secreted protein since the protein contains a putative signal peptide. Methods: Initially, four different promoters (P43, Pspac, PxylA, and PyxiE) were compared for their strength using GFP as a reporter in B. subtilis. Subsequently, six different signal peptides (SacB, Epr, AmyE, AprE, LipA, and Vip3A)were tested in conjunction with the selected promoter and mtx2 to evaluate levels of Mtx2 secreted by B. subtilis WB800, an extracellular protease-deficient strain. Results: The promoter PyxiE showed the highest GFP intensity and was selected for further study. Mtx2 was successfully produced as a secreted protein from signal peptides LipA and AmyE, and exhibited larvicidal activity against Aedesaegypti. Conclusion: B. subtilis was successfully developed as a host for the production of secreted Mtx2 and the protein retained its larvicidal activity. Although the Mtx2 production level still needs improvement, the constructed plasmids could be used to produce other soluble proteins.

scholarly journals Genetic Manipulation Of Tomato (Lycopersicon Esculentum) Using Wga Gene Through Agrobacterium Mediated Transformation

1970 ◽  
Vol 8 (1) ◽  
pp. 36-43 ◽  
Author(s):  
SL Shantha ◽  
M Padma ◽  
SG Bhat ◽  
C Sunil Kumar ◽  
B Ragavendra Rao
Keyword(s):  
Genetic Manipulation ◽  
Kanamycin Resistance ◽  
Direct Regeneration ◽  
Pcr Analysis ◽  
Leaf Discs ◽  
Pollution Levels ◽  
Plant Parts ◽  
Recombinant Vector ◽  
Resistance To Insects ◽  
Cetyl Trimethyl Ammonium

The major problems faced in the field of Agriculture are loss in crop yield caused by insects, Herbs (weeds), viruses and the pathogens and the pests associated loss is about 14% of total agricultural production. The use of pesticides has resulted in adverse effect on the beneficial organisms and other plant parts such as leaves, fruits and has reached pollution levels, which has become a major concern for environmentalists. Therefore, products of crops resistant to insects have been the first priority in crop biotechnology. Genetic transformation has led the possibility of transforming crops for enhanced resistance to insects through the use of insect control protein gene-WGA (Wheat Germ Agglutinin), a glycoprotein with molecular weight of 34000 Da. Toxic effect appears to be mediated through binding of the lectins (WGA) to glycoproteins in the insect leading to the disruption of gut epithelial cells and are believed to be “natures own insecticides”. The present study involves preparation of recombinant pGPTV vector having WGA gene, which was transferred to E. coli DH5α basic strain. The recombinant vector was transferred to Agrobacterium tumefaciens strain LBA4404 using helper strain through triparental mating. The recombinant vector having Agrobacterium was infected with tomato leaf discs through co-cultivation and the leaf discs were transferred to selection media containing Kanamycin and direct regeneration of the plantlet were obtained from the leaf discs. The npt-II gene (Kanamycin resistance gene) serves as a selectable marker system in plants. The regenerated plantlets grown on selection media was subjected to primary screening by isolating the genomic DNA by CTAB (Cetyl Trimethyl Ammonium Buffer) method and the transformation was confirmed by the presence of amplified fragments of WGA gene by PCR analysis. DOI: http://dx.doi.org/10.3126/kuset.v8i1.6041 KUSET 2012; 8(1): 36-43

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