Team:BIT-China/Promoter
Lambda Red recombination
Background
Recombineering (recombination-mediated genetic engineering) is an efficient molecular engineering technique used for gene replacement, deletion and insertion. Compared to the traditional technology like digestion and ligation, it’s based on the homologous recombination (HR) and can achieve DNA modification without the restriction enzyme and ligase.
The homologous recombination in E.coli is mediated by proteins called recombinase. The traditional way is dependent on the RecA protein. Nevertheless, there are some limitations like the requirements for long homologous region and the low efficiency of successful recombination. In 1998, a new technique based on Red recombination system is firstly reported to dramatically improve the capacity of gene replacement on the chromosome of E.coli [1]. In recent decades, Red recombination has been extensively employed in gene editing of E.coli due to its distinguished advantages, such as the high efficiency of recombination and the requirement of short homologous arms.
The homologous recombination in E.coli is mediated by proteins called recombinase. The traditional way is dependent on the RecA protein. Nevertheless, there are some limitations like the requirements for long homologous region and the low efficiency of successful recombination. In 1998, a new technique based on Red recombination system is firstly reported to dramatically improve the capacity of gene replacement on the chromosome of E.coli [1]. In recent decades, Red recombination has been extensively employed in gene editing of E.coli due to its distinguished advantages, such as the high efficiency of recombination and the requirement of short homologous arms.
Mechanism
Cloning methods based on cleavage and joining reactions are used for DNA modification and amplification on plasmid vectors, but it’s limited to the gene editing on chromosome. According to the DNA double strand break and repair recombination pathway, the modified tool—“Red recombination” was created for efficient operation on chromosome [2].
To generate recombinant DNA molecules and achieve gene replacement, three steps are required. Firstly, primers containing 30-50 nt sequence identical to the target sequence are synthesized to attain the PCR product homologous with the target. Second, cells are induced for Exo, Beta and Gam function and are made competent for electroporation. Finally, recombination occurs between the amplified PCR product and the target site on chromosome after electroporation [3].
To generate recombinant DNA molecules and achieve gene replacement, three steps are required. Firstly, primers containing 30-50 nt sequence identical to the target sequence are synthesized to attain the PCR product homologous with the target. Second, cells are induced for Exo, Beta and Gam function and are made competent for electroporation. Finally, recombination occurs between the amplified PCR product and the target site on chromosome after electroporation [3].
Application in our project
The phenomenon of plasmid lost is very common. To prove a new concept of plasmid quorum sensing, we need to adjust our design according to different situations. Typically, the losing of plasmid will happen both in low-copy plasmid and high-copy one. Since our gene circuits for sensing and controlling the plasmid numbers are also loaded on the target plasmid, we need to avoid the completely lost of plasmids or the system will not reach the expected effects. That is, the engineered E.coli cannot sense and control the plasmids due to the deficiency of its functional parts.
To solve the problem above, we aim to employ the Lambda Red Recombination system to integrate the functional parts of our project into the genome. The two main factors which will influence the recombination efficiency are the target sites and the length of homologous arms.
After a period of try-error, we have successfully integrate several functional circuits in the form of DNA linear fragments into the chromosome of E.coli DH5α. The efficient tool of genome editing based on recombination has expanded the application of our project and greatly facilitated the measurement of our setting thresholds when the plasmid comes to the low-copy concentration.
To solve the problem above, we aim to employ the Lambda Red Recombination system to integrate the functional parts of our project into the genome. The two main factors which will influence the recombination efficiency are the target sites and the length of homologous arms.
After a period of try-error, we have successfully integrate several functional circuits in the form of DNA linear fragments into the chromosome of E.coli DH5α. The efficient tool of genome editing based on recombination has expanded the application of our project and greatly facilitated the measurement of our setting thresholds when the plasmid comes to the low-copy concentration.
REFERENCE
[1] Murphy K C. Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. [J]. Journal of Bacteriology, 1998, 180(8):2063-71.
[2] Yu D, Ellis H M, Lee E C, et al. An efficient recombination system for chromosome engineering in Escherichia coli. [J]. Proceedings of the National Academy of Sciences, 2000, 97(11):5978-5983.
[3] Lynn T, Court D L, Mikail B, et al. Recombineering: genetic engineering in bacteria using homologous recombination. [J]. 2014, 106: Unit 1.16-Unit 1.16.
[1] Murphy K C. Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. [J]. Journal of Bacteriology, 1998, 180(8):2063-71.
[2] Yu D, Ellis H M, Lee E C, et al. An efficient recombination system for chromosome engineering in Escherichia coli. [J]. Proceedings of the National Academy of Sciences, 2000, 97(11):5978-5983.
[3] Lynn T, Court D L, Mikail B, et al. Recombineering: genetic engineering in bacteria using homologous recombination. [J]. 2014, 106: Unit 1.16-Unit 1.16.
