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The Thermo Scientific aLICator™ LIC Cloning and Expression System is designed for fast and efficient ligation independent cloning and tight regulation of gene expression in E. coli. The pLATE bacterial expression vectors are designed for high levels of target protein expression in concert with minimal background (uninduced) expression, which permits expression of proteins that are toxic to E. coli cells. To streamline and facilitate the process of insert cloning into the expression vector, the aLICator system uses directional LIC cloning technology, a rapid procedure that provides high-cloning efficiencies.
The tightly regulated expression and fast, efficient directional cloning makes the aLICator LIC Cloning and Expression System the best choice for routine and toxic gene cloning and expression in E. coli.
The aLICator LIC cloning system uses directional LIC cloning technology to streamline and facilitate cloning into an expression vector. LIC ensures high-cloning efficiencies of more than 95% and eliminates the need for ligation and restriction enzyme digestion steps.
The LIC method uses T4 DNA polymerase to create specific 14 to 21 nucleotide single-stranded overhangs on the pLATE vectors and DNA inserts (see Reference 2). 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 2). After annealing, the LIC vector and insert are transformed into competent E. coli cells without the use of T4 DNA ligase. Covalent bond formation at the vector-insert junctions occurs within the cell to yield circular plasmid.
The system consists of four kits 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 clone into all three vectors and determine the most compatible vector for further research.
Kit components were functionally tested in control experiments as outlined in the manual. A 2.5 µL aliquot of the LIC mixture was used to transform 50 µL of chemically competent XL1-Blue cells of > 106 cfu/µg DNA transformation efficiency.
Cloning efficiency of the control PCR product into the LIC vector was > 4 x 104 cfu/µg DNA and > 95% of the recombinant plasmids contained the appropriate insert.
Figure 1 | 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.
Studies have shown that expression of T7 RNA polymerase from the lacUV5 promoter in λDE3 lysogens is leaky, even in the absence of inducer (1). If the gene of interest is toxic to the E. coli host, basal expression of the gene may lead to plasmid instability and/or cell death. To overcome this problem pLATE vectors contain additional elements that reduce basal T7 RNA polymerase expression 2 – 2.5 fold. These elements include 2 lac operators that flank the T7 promoter and serve as binding sites for the lac repressor (encoded by the lacI gene). A rrnB1-B2 terminator is also placed upstream of the cloning site to prevent basal gene expression from vector derived promoter-like elements. In addition, a constitutively induced weak Tet promoter (Ptet) operates in the opposite direction to the T7 promoter, further reducing basal expression.
Target genes are directionally cloned into the pre-linearized pLATE vectors. Following protein affinity purification, amino-terminal tags can be removed via enterokinase (DDDDK^) protease cleavage sites located immediately N-terminal to the target protein.
Inserts can be conveniently subcloned using rare-cutting restriction enzymes via recognition sequences present on both sides of the LIC cloning site.
Restriction endonuclease Cfr9I R gene was cloned into pLATE expression vectors and transformed into E.coli DH10B cells having Cfr9I methylase (without induction). Then the plasmid constructs after sequence verification were re-transformed into expression strain ER2566 and cultivated under induction conditions (1 mM IPTG). After 3 hours post induction bacterial cells were collected, normalized according optical density and sonicated. 1 µl of cell free extract was assessed for Cfr9I restriction activity by ability to cleave Lambda DNA. M - GeneRuler High Range DNA Ladder (#SM1353), 1 - Lambda DNA, 2 - Lambda DNA / Cfr9I, 3 - 6 - cell free extract with Cfr9I expressed within pLATE11, pLATE31, pLATE51 and pLATE52 vectors respectively.
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C. Aslanidis, P. J. de Jong, Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18(20), 6069–6074 (1990).
K. N. Rand, Crystal Violet can be used to Visualize DNA Bands during Gel Electrophoresis and to Improve Cloning Efficiency. Elsevier Trends Journals Technical Tips. Online, T40022 (1996).
S. Adkins, M. Burmeister, Visualization of DNA in agarose gels and educational demonstrations. Anal Biochem. 240(1), 17-23, (1996).