scholarly journals Rapid and Efficient BAC Recombineering: Gain & Loss Screening System

2020 ◽  
Author(s):  
Myeong Uk Kuk ◽  
Sekyung Oh ◽  
Joon Tae Park
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Screening Method ◽  
Artificial Chromosome ◽  
Screening Strategy ◽  
Limited Time ◽  
Screening System ◽  
Time Period ◽  
New Strategy ◽  
Gain Loss ◽  
Entire Procedure

AbstractRecombineering has been developed to modify bacterial artificial chromosome (BAC) via homologous recombination. Nevertheless, as a screening strategy to identify the correct clone was not properly developed, it was difficult to obtain a correct clone within a limited time period. To address these issues, we developed a new screening method (a gain & loss screening system) that enables the efficient identification of the recombineered clone. Simple inoculation of cells into LB medium with appropriate antibiotics visually revealed the positive clones within 24 h. DNA sequencing confirmed 100% accuracy of this screening method by showing that all positive clones exhibited recombinant sequences. Furthermore, our new method allowed us to complete the entire procedure consisting of 1st recombineering, flip-out and 2nd recombineering in just 13 days. Overall, our new strategy may provide a new avenue for BAC recombeerining, as evidenced by markedly increased accuracy and subsequently shortened recombineering duration.

Rapid and Efficient BAC Recombineering: Gain & Loss Screening System

2020 ◽  
Author(s):  
Myeong Uk Kuk ◽  
Sekyung Oh ◽  
Joon Tae Park
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Homologous Recombination ◽  
Screening Method ◽  
Artificial Chromosome ◽  
Screening Strategy ◽  
Limited Time ◽  
Screening System ◽  
Time Period ◽  
New Strategy ◽  
Entire Procedure

Abstract Background Recombineering has been developed to modify bacterial artificial chromosome (BAC) via homologous recombination. Nevertheless, as a screening strategy to identify the correct clone was not properly developed, it was difficult to obtain a correct clone within a limited time period. To address these issues, we developed a new screening method (a gain & loss screening system) that enables the efficient identification of the recombineered clone.Results Simple inoculation of cells into LB medium with appropriate antibiotics visually revealed the positive clones within 24 h. DNA sequencing confirmed 100% accuracy of this screening method by showing that all positive clones exhibited recombinant sequences. Furthermore, our new method allowed us to complete the entire procedure consisting of 1st recombineering, flip-out and 2nd recombineering in just 13 days.Conclusion Overall, our new strategy may provide a new avenue for BAC recombeerining, as evidenced by markedly increased accuracy and subsequently shortened recombineering duration.

scholarly journals Targeting CreERT2 expression to keratin 8-expressing murine simple epithelia using bacterial artificial chromosome transgenesis

2012 ◽  
Vol 21 (5) ◽  
pp. 1117-1123 ◽  
Author(s):  
Li Zhang ◽  
Boyu Zhang ◽  
Sang Jun Han ◽  
Amy N. Shore ◽  
Jeffrey M. Rosen ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Artificial Chromosome ◽  
Keratin 8

scholarly journals Revising the Chromosome-Specific Probes of White Hawk (Leucopternis albicollis)

Proceedings ◽  
2020 ◽  
Vol 76 (1) ◽  
pp. 1
Author(s):  
Ivanete Furo ◽  
Rafael Kretschmer ◽  
Jorge Pereira ◽  
Darren Griffin ◽  
Rebecca O’Connor ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Artificial Chromosome ◽  
Bird Of Prey ◽  
Inaccurate Data ◽  
Intrachromosomal Rearrangements

Leucopternis albicollis is a diurnal bird of prey with extensive karyotype reorganization. Chromosome-specific probes from this species have been used successfully to detect intrachromosomal rearrangements in different species of bird since 2010. However, some gaps were detected in this first set of probes. Here, we have obtained a new set of whole chromosome probes in order to improve the previous one; also, we have performed experiments using bacterial artificial chromosome (BAC) from chicken microchromosomes. Our results demonstrated that the microchromosomes were involved in fusion events. In addition, a new nomenclature has been proposed for the new set of probes and some inaccurate data were corrected.

scholarly journals A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

2021 ◽  
Vol 22 (6) ◽  
pp. 3041
Author(s):  
Gheorghita Menghiu ◽  
Vasile Ostafe ◽  
Radivoje Prodanović ◽  
Rainer Fischer ◽  
Raluca Ostafe
Keyword(s):  
High Throughput ◽  
Cell Sorting ◽  
High Throughput Screening ◽  
Screening Method ◽  
Enzymatic Reaction ◽  
Hydrolytic Enzymes ◽  
Screening System ◽  
Fluorescence Activated Cell Sorting ◽  
Fungal Cell ◽  
Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

Construction and initial analysis of bacterial artificial chromosome (BAC) contigs from the sex-determining region of the platyfish Xiphophorus maculatus

Gene ◽  
2002 ◽  
Vol 295 (2) ◽  
pp. 247-254 ◽  
Author(s):  
Alexander Froschauer ◽  
Cornelia Körting ◽  
Takayuki Katagiri ◽  
Takashi Aoki ◽  
Shuichi Asakawa ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Artificial Chromosome ◽  
Initial Analysis ◽  
Xiphophorus Maculatus ◽  
Bac Contigs
Sign in / Sign up

Export Citation Format

Share Document