The traditional methods of inserting genes into vectors are based on DNA cleavage by restriction endonuclease and then ligation by DNA ligase. However, this approach to DNA engineering is time-consuming and relatively inefficient. Ligation Independent Cloning (LIC) technique is developed as an alternative to restriction endonuclease/ligase cloning. LIC provides a simple and cost-effective tool for producing many constructs of a single target or multiple targets in parallel without the need to select specific restriction enzymes for each gene.
T4 DNA polymerase is a template-dependent DNA polymerase that has 3’-exodeoxyribonuclease activity, but lacks 5’ to 3’ exodeoxyribonuclease activity. LIC uses the 3´-exodeoxyribonuclease activity of T4 DNA polymerase to assemble DNA molecules by non-covalent complementation of single-stranded DNA of the insert and vector. Briefly, LIC relies on about 10- to 15-nucleotide complementary 3′ overhangs at the ends of a PCR-amplified DNA fragment and a linearized vector to make a stable hybridization product that can be readily used to transform host organisms without ligation. LIC based on T4 DNA polymerase cloning is strictly sequence-dependent as it requires the presence or absence of specific nucleotides at certain locations in the overlapping region.
pColiExpress™ LIC Cloning & Expression Kits are a highly efficient, versatile and fast system of DNA cloning vectors for protein expression in E. coli. All family systems are based in directional LIC technology, a rapid procedure that provides high-cloning efficiency and the fast production of a large quantity of any desired proteins.
Product Name | pColiExpress™ I | pColiExpress™ II | pColiExpress™ III | pColiExpress™ IV | |
---|---|---|---|---|---|
Catalog Number | BE001 | BE005 | BE010 | BE015 | |
Features | |||||
5'His6 Tag | √ | ||||
3'His6 Tag | √ | √ | √ | ||
Periplasmic expression | √ | ||||
5'cmyc Tag | √ | ||||
HRV protease cut site | √ | ||||
Resistance Marker-Ampicillin | √ | √ | √ | √ | |
T7 promoter | √ | √ | √ | √ | |
Low copy number (ori pBR322) | √ | √ | √ | √ | |
Strain Protein Expression |
BL21 (DE3) | √ | √ | √ | √ |
BL21 (DE3) pLys | √ | √ | √ | √ |
Product Name | Applications |
---|---|
pColiExpress™ I Cloning & Expression Kit |
|
pColiExpress™ II Cloning & Expression Kit | |
pColiExpress™ III Cloning & Expression Kit | |
pColiExpress™ IV Cloning & Expression Kit |
Product Name | Applications |
---|---|
BL21 (DE3) Competent Cells | Protein Expression using the T7 Expression System. |
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