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 competent E. 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.
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^.
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 and Expression Kits||aLICator LIC Cloning and Expression Kit 1 (untagged)||pLATE 11||K1241||20 rxns|
|aLICator LIC Cloning and Expression Kit 2|
|pLATE 51||K1251||20 rxns|
|aLICator LIC Cloning and Expression Kit 3|
|pLATE 31||K1261||20 rxns|
|aLICator LIC Cloning and Expression Kit 4|
|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|
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For Research Use Only. Not for use in diagnostic procedures.