Reliable Scarless Fusion of Genes through Overlap Extension PCR to Develop Chimeric Genes

AbstractObjective:Restoration of catalytic activity of Isa2 fromMethods:The six conserved amino acid residues absent in the Stisa2 gene were restored by mutation using the overlap extension PCR and the asymmetrical overlap extension PCR methods. Next, mutant Stisa2 with restored catalytic residues was expressed inResults:Both qualitative and quantitative analysis showed that the restoration of the conserved residues in the catalytic site did not restore starch debranching activity. Molecular modeling showed greater than expected distances between the catalytic triad in mutant Stisa2. These additional distances are likely to prevent hydrogen bonding which stabilizes the reaction intermediate, and are critical for catalytic activity.Conclusions:These results suggest that during evolution, mutations in other highly conserved regions have caused significant changes to the structure and function of the catalytic network. Catalytically inactive Isa2, which is conserved in starch-producing plants, has evolved important non-catalytic roles such as in substrate binding and in regulating isoamylase activity.

Download Full-text

Overlap Extension PCR Cloning

Materials and Methods ◽

10.13070/mm.en.1.43 ◽

2011 ◽

Vol 1 ◽

Author(s):

Mary Johnson

Keyword(s):

Pcr Cloning ◽

Overlap Extension Pcr ◽

Overlap Extension

Download Full-text

Synthesis, cloning, and expression of Mycoplasma suis inorganic pyrophosphatase gene using PCR-based accurate synthesis and overlap-extension PCR, and its immunogenicity analysis

Research in Veterinary Science ◽

10.1016/j.rvsc.2011.02.009 ◽

2011 ◽

Vol 91 (3) ◽

pp. e100-e102 ◽

Cited By ~ 4

Author(s):

Jianzhu Liu ◽

Ziqiang Cheng ◽

Dong Zhou ◽

Li Zhang ◽

Zhengui Yan ◽

...

Keyword(s):

Cloning And Expression ◽

Inorganic Pyrophosphatase ◽

Overlap Extension Pcr ◽

Mycoplasma Suis ◽

Overlap Extension

Download Full-text

A Short Protocol for Gene Knockout and Complementation in Xylella fastidiosa Shows that One of the Type IV Pilin Paralogs (PD1926) Is Needed for Twitching while Another (PD1924) Affects Pilus Number and Location

Applied and Environmental Microbiology ◽

10.1128/aem.01167-18 ◽

2018 ◽

Vol 84 (18) ◽

Cited By ~ 7

Author(s):

Prem P. Kandel ◽

Hongyu Chen ◽

Leonardo De La Fuente

Keyword(s):

Natural Transformation ◽

Gene Knockout ◽

Xylella Fastidiosa ◽

Twitching Motility ◽

Wild Type ◽

Content Type ◽

Overlap Extension Pcr ◽

Type Iv ◽

Overlap Extension ◽

Pilin Subunit

ABSTRACT Twitching motility is one of the major virulence factors of the plant-pathogenic bacterium Xylella fastidiosa, and it is mediated by type IV pili (TFP) that are present at one of the cell poles. Genome analysis of X. fastidiosa showed the presence of at least four paralogs of the gene pilA, which encodes the TFP major pilin subunit. However, whether all of these paralogs have a functional role in TFP structure and function is unknown. Here, using a short and reliable protocol based on overlap extension PCR and natural transformation, deletion mutants of two pilA paralogs (pilA1 PD1924 and pilA2 PD1926) were generated in two X. fastidiosa subsp. fastidiosa strains, WM1-1 and TemeculaL, followed by assessment of twitching motility and biofilm formation. Deletion of pilA2 caused loss of twitching motility, whereas deletion of pilA1 did not influence twitching motility but caused hyperpiliation and extended distribution of TFP along the sides of the cell. Loss of twitching motility due to pilA2 deletion was restored when a wild-type copy of the pilA2 gene was added at a neutral site in the genome of mutants in both wild-type backgrounds. This study demonstrates that PCR templates generated by overlap extension PCR can be successfully used to rapidly generate gene knockouts and perform genetic complementation in X. fastidiosa, and that twitching motility in X. fastidiosa is controlled by regulating the transcription of the major pilin subunit, pilA2. IMPORTANCE The bacterial plant pathogen Xylella fastidiosa causes incurable diseases in multiple hosts, including grape, citrus, and blueberry. Historically restricted to the Americas, it was recently found to cause epidemics in olives in Italy and to infect other hosts in Europe and Asia. In this study, we report a short protocol to create deletion and complemented mutants using fusion PCR and natural transformation. We also determined the distinct function of two pilin paralogs, the main structural component of TFP involved in twitching motility, which allows this bacterium to move inside the xylem vessels against the flow. One of the paralogs is needed for twitching movement, whereas the other does not have an effect on motility but influences the number and position of TFP. Since twitching motility is fundamental for the virulence of this xylem-limited bacterium, this study contributes to the understanding of the regulation of virulence by this pathogen.

Download Full-text