Part:BBa_K3333007
lambda-red (Gam-Beta-Exo)
Lambda-Red is a recombinase system, which originated from the bacteriophage λ. This part can mediate recombination for multiplex genome editing.
Usage and Biology
This part includes three genes, γ, β, and exo, these genes respectively encode protein Gam, Bet, and Exo [1]. λ-red system is a widely used recombination system deriving from phage λ, containing three proteins: Exo, Beta and Gam. This system can help recombine dsDNA/ssDNA into a new DNA molecule, serving as a useful and practical tool in many other projects. There are currently three models explaining the elaborate mechanisms of the λ-red system: RecA-dependent, ssDNA annealing (RecA-independent) and the recent Replisome Invasion/Template Switch model[2]. What makes the λ-red system stands out is its high efficiency compared to restriction enzymes as well as its ability to promote recombination events between DNA species with as little as 40bp of shared sequence at high frequency.[3] It is also worth mentioning that recombinant formation promoted by the λ-red system is IPTG dependent, supported by the fact that longer exposure to IPTG results in greater amounts of recombinants per competent cell.[4]
λ Exo, working as an exonuclease with a preference for 5’-phosphates, degrades the 5’-ended strand dsDNA and thus creating a 3’-ended overhang. This exonuclease exists as a trimer in a toroidal shape with a funnel-shaped central channel. It is presumed that the dsDNA passes through the central channel and is then acted upon the by one of the three acting sites and finally exits as ssDNA.[2] Particularly, the side chain of Leu78 inserts between the second and third bases of the 5’-ended strand so as to separate the two terminal nucleotides in the active site.[5] Besides, in the active site to which the DNA is bound, there are two Mg2+ ions showing octahedral coordination, revealing that λ exonuclease uses a classic two-metal mechanism which is also seen in several TypeⅡrestriction endonucleases.[5]
λ Beta is a protein capable of meditating strand annealing and promoting the recombination of complementary strands. Beta exists in three structural states: small rings, large rings and helical filaments. When encountered with DNA, large beta rings bind ssDNA and therefore initiate the annealing with a complementary strand. The annealing process continues spontaneously to generate a dsDNA supercoiled within the β helical filament [6]. Beta binds to ssDNA and delivers ssDNA to the target replication fork, the Bet bound ssDNA behaves like Okazaki fragment and is introduced into the genome[1]
λ Gam, also known as the anti-RecBCD protein, functions by directing binding to the DNA-binding site of RecBCD and subsequently inhibiting RecBCD’s capability to bind to dsDNA ends. This protein is an all helical dimeric structure with two hydrophobic N-terminal H1 helices sticking out from a dimerization domain. The Gam dimerization domain is proposed to be a double-stranded DNA mimetic while helix H1 may mimic ssDNA. [7] These two helices are speculated to be inserted into channels within the RecB and RecC subunits which were originally occupied by 3’-ssDNA and 5’-ssDNA of an unwound dsDNA substrates. Besides, aromatic residues are assumed to interact with the bases of the ssDNA.[2]
In order to produce the engineered phages, we constructed two plasmids, one carrying Cas9 and Lambda-Red, while the other one carrying the TA system genes. Cas9 system and Lambda-Red system is associated to edit the natural phages vB_PaeM_SCUT-S1, to make it carry the toxin gene. We are hoping to utilize this λ-red system to insert a toxin gene into the phage genome together with the CRISPR/Cas9 system. As a result, this phage will acquire the ability to cause excessive toxin expression in the host cell after infection.
Reference
[1]Jeong, J., Cho, N., Jung, D. & Bang, D. Genome-scale genetic engineering in Escherichia coli. Biotechnol Adv 31, 804-810, doi:10.1016/j.biotechadv.2013.04.003 (2013).
[2]Court R, Cook N, Saikrishnan K, Wigley D (2007). The crystal structure of lambda-Gam protein suggests a model for RecBCD inhibition. JOURNAL OF MOLECULAR BIOLOGY 371: 25-33.
[3]Murphy KC (1998). Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. JOURNAL OF BACTERIOLOGY 180: 2063-2071.
[4]Murphy KC (2016). lambda Recombination and Recombineering. EcoSal Plus 7.
[5]Passy SI, Yu XN, Li ZF, Radding CM, Egelman EH (1999). Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 96: 4279-4284.
[6]Poteete AR (2001). What makes the bacteriophage lambda Red system useful for genetic engineering: molecular mechanism and biological function. FEMS MICROBIOLOGY LETTERS 201: 9-14.
[7]Zhang J, McCabe KA, Bell CE (2011). Crystal structures of lambda exonuclease in complex with DNA suggest an electrostatic ratchet mechanism for processivity. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 108: 11872-11877.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1481
Illegal PstI site found at 976
Illegal PstI site found at 1223 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1481
Illegal PstI site found at 976
Illegal PstI site found at 1223 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1481
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1481
Illegal PstI site found at 976
Illegal PstI site found at 1223 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1481
Illegal PstI site found at 976
Illegal PstI site found at 1223
Illegal AgeI site found at 1403 - 1000COMPATIBLE WITH RFC[1000]
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