Ligation Independent Cloning (LIC) is an alternative to restriction enzyme/ligase cloning and ensures high cloning efficiencies of more than 95%.

The LIC method uses T4 DNA polymerase to create specific 14 to 21 nucleotide single-stranded overhangs on vector and DNA insert. T4 DNA polymerase has two enzymatic activities: 5'→3' polymerase activity and 3'→5' exonuclease activity. The exonuclease activity removes nucleotides from the 3' ends of the DNA while the polymerase activity restores the chain using dNTPs and the complementary DNA strand as a template. In the LIC protocol, only dGTP is included in the reaction, causing the 3'→5'-exonuclease and 5'→3'-polymerase activities to equilibrate at the first occurrence of cytosine in the complementary strand (see Figure 1). After annealing, the LIC vector and insert are transformed into competentE. coli cells. Covalent bond formation at the vector-insert junctions occurs within the cell to yield circular plasmid.

Figure 1: Generation of sticky ends on the gene of interest with T4 DNA polymerase and dGTP

Successful cloning & robust protein expression

The Thermo Scientific aLICator LIC Cloning and Expression System is designed for fast and efficient LIC cloning and subsequent tight regulation of gene expression in E. coli.

pLATE bacterial expression vectors (see Figure 2) are designed for high levels of target protein expression in concert with minimized basal (uninduced) expression.

The aLICator system consists of 4 kits and 2 sets, based on the pLATE series of bacterial expression vectors. For proteins with a known preference for either the N- or C-terminal 6xHis-tag position, using the appropriate N- or C-terminal kit is recommended. When the protein structure and features are not well known, it is recommended to use an aLICator set. Sets allow cloning into three different vectors to determine the most compatible vector for further experiments. Following protein affinity purification, aminoterminal tags can be removed either via Enterokinase (EK), DDDDK^ or a novel protease: WELQut (WQ), WELQ^.


Figure 2:  pLATE expression vectors use elements from bacteriophage T7 to control expression of heterologous genes in E. coli.
The expression of the gene of interest is driven by a strong bacteriophage T7 promoter that is specifically recognized by T7 RNA polymerase. To express the gene of interest, E. coli strains such as BL21 (DE3), HMS 174 (DE3) must be used, in which expression of T7 RNA polymerase gene is under the control of an inducible promoter such as lacUV5. After IPTG induction, theT7 RNA polymerase is expressed within the cell, and begins transcription of genes under the T7 promoter.

Click image to enlarge.

 

aLICator highlights

  • High efficiency LIC cloning
  • Tight control of gene expression
  • High yield expression
  • Versatile – tagged or untaged protein expression with tag removal option

aLICator applications

  • Directional PCR product cloning
  • Tightly regulated protein expression
  • Expression of toxic genes

Expression of toxic genes with aLICator

Tightly regulated gene expression in pLATE vectors allows for cloning of toxic genes in E. coli cells.

As an example, the toxic restriction endonuclease Cfr9I R gene was cloned into pLATE expression vectors and transformed into E.coli DH10B cells having Cfr9I methylase (without induction). The plasmid constructs were re-transformed into expression strain ER2566 and cultivated under induction conditions (1 mM IPTG). 3 hours post induction, bacterial cells were collected, normalized according to optical density and sonicated. 1 µL of cell free extract was assessed for Cfr9I activity through digestion of lambda DNA (see lanes 3-6 below).

M – Thermo Scientific GeneRuler High Range DNA Ladder (#SM1353)
1 – Undigested lambda DNA
2 – Cfr91 digested lambda DNA
3, 4, 5, 6 – Lambda DNA mixed with cell free extract with Cfr91 expressed from pLATE11, pLATE31, pLATE51 and pLATE52 vectors respectively.

aLICator LIC cloning products

  Product Vector Catalog # Size
aLICator LIC Cloning and Expression Kits aLICator LIC Cloning and Expression Kit 1 (untagged) pLATE 11 K1241 20 rxns
aLICator LIC Cloning and Expression Kit 2
(N-terminal His-tag/EK)
pLATE 51 K1251 20 rxns
aLICator LIC Cloning and Expression Kit 3
(C-terminal His-tag)
pLATE 31 K1261 20 rxns
aLICator LIC Cloning and Expression Kit 4
(N-terminal His-tag/WQ)
pLATE 52 K1281 20 rxns
aLICator LIC Cloning and Expression Sets aLICator LIC Cloning and Expression Set 1 (All-in-One/EK) pLATE 11, 31, & 51 K1271 30 rxns
aLICator LIC Cloning and Expression Set 2 (All-in-One/WQ) pLATE 11, 31, & 52 K1291 30 rxns
  WELQut Protease EO0861 500 U

Vector sequences

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pLATE bacterial expression vector sequences

Control PCR fragments