pXST, a novel vector for TA cloning and blunt-end cloning

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Advancements in sequencing technologies generate vast genomic data.
• A need exists for cost-effective, rapid, and efficient cloning vectors for large-scale gene cloning and functional studies.

Purpose of the Study:

• To construct and validate a novel bifunctional cloning vector, pXST.
• To assess the efficiency of pXST for TA cloning and blunt-end ligation of PCR-amplified cassava genomic fragments.

Main Methods:

• Construction of the pXST vector featuring XcmI-ccdB-XcmI cassette and SmaI restriction site.
• Cloning of PCR-amplified cassava DNA fragments using both TA cloning and blunt-end ligation into the pXST vector.
• Evaluation of transformation efficiency and positive cloning efficiency for different insert sizes.

Main Results:

• The pXST vector successfully facilitated both TA cloning and blunt-end ligation.
• TA cloning generally yielded higher transformation efficiency than blunt-end ligation, especially for smaller inserts.
• The highest transformation efficiency of 8.6 × 10^6 transformants/μg was achieved with a 517 bp fragment using TA cloning.
• Positive cloning efficiency reached up to 100%, particularly for small inserts, with no significant difference between ligation methods.

Conclusions:

• The pXST vector is a versatile tool for gene cloning, supporting both TA and blunt-end ligation.
• The vector enables direct selection of positive recombinants in E. coli via ccdB gene disruption.
• pXST offers a high-performance, cost-effective alternative for gene cloning experiments.