Team:ZJU-China/SSR

Abstract

Lambda red is a Lambda phage derived recombination system which contains three proteins: Exo, Beta and Gam. It can recombine dsDNA/ssDNA into different kinds of DNA molecules such as chromosome, bacteria artificial chromosome (BAC) or even multicopy plasmids, as long as each side of the donor dsDNA/ssDNA is flanked by 36-50bp homologous arms^[1]. The homologous arms also determine the precise sites of recombination, thus the donor fragment can be inserted into any sites by choosing and adding homologous arms using PCR.

Lambda red has been shown to be a reliable and versatile recombination method. Here we will introduce you the brief mechanism of Lambda red and some current strategies in genetic engineering (so called "recombineering"). You will see that our GeneSocket system not only utilize all the advantages of Lambda red systems but also change the whole technology to make recobineering simpler and faster.

Lambda red composition

• Exo:
  

  Exo is a 5’→3’ double-strand DNA specific exonuclease and is only required for dsDNA recombination. It can degrade one of the whole strand of dsDNA to allow a single strand annealing recombination^[2].

• Beta:
  

  Beta is a single strand binding protein, which is the central player in red system for both dsDNA and ssDNA recombination. It binds and aims the donor fragment to homologous sites.

• Gam:
  

  The Gam protein inhibits the E. coli RecBCD exonuclease that normally degrades all linear dsDNA. It is important to use dsDNA as donor fragments.

• Homologous arm

  Homologous arms are short sequences flanking each side of the donor sequence. It also matches the recombination sites on the acceptor region. For high possibility of recombination, it may be better if the homologous arms are longer, but actually 36-50bp are enough for antibiotic resistance screening as most researchers has been reported^[1].

  Table.1 Allele designations of chromosomal gene disruptions1

  The sequences of homologous arm are not limited by specific recombination sites like the demand of many other recombinase, but there are also some factors we need to consider when choosing the arms. Fortunately, ZJU-China has design a tool to help you carry out this process easily! For more information, please see our "GS-BOX".

Importantly, host recombination functions are not needed in red recombination, including a key endogenic recombination protein RecA^[3]. Therefore, Lambda red system is a flexible and simple system that can be carried on a plasmid. For our GeneSocket, we call this plasmid as "Support device".

Ability and normal application of Lambda red systems

Lambda red based recombineering can make gene replacements, deletions, insertions, inversions, and single and multiple point mutations. It’s a kind of mature method. Some people have used it to make construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection, which use the same resistance gene to recombine chromosome regions rank from 20 to 7000bp^[4]. Current strategies of using Lambda red are reliable but mainly focus on single changes. If we want to do multiple changes on chromosome as the need of synthetic biology, we must try to find a new better way.

Here is one of the current strategies using Lambda red^[5]:

With this strategy, we have at least transformed two plasmids and done negative selection twice. See the page to know the difference between GeneSocket method and current strategies. You’ll find which one is faster and easier.

Figure.4 λ-red current method	Preparation 1.Genes of interest (GOI) are going to be inserted. 2.Insertion sites should be found, a series of plasmids are used to acquire resistance gene with FRT or LoxP sites. 3.Fragments are prepared by PCR and ligation.
	Recombination 4.Insertion mode: GOI is linked to a resistance gene, and two recombination sites flank each side. 5.Plasmids like pKD46 carrying Lambda red genes to allow recombination to happen.
	Resistance gene retrieve 6.pKD46 should be discard after this step, and a new plasmid like pCP20 carry recombinase is transformed into cell to retrieve resistance gene. 7.Scar cannot be avoided by this method for the use of recombinase sites.

Summary

Lambda red is a broadly used recombination system, and recombineering based on Lambda red also shows large protential in chromosome engineering. Generally, current recombineering strategies mainly focus on inactivating or inserting a single gene, so although the protocol are quite complicated, it is acceptable. If we want to do continuous recombination round by round, the current strategies may have trouble doing this. Therefore, it is necessary to change the work flow of recombination fundamentally. Later you will see that our GeneSocket build recombination & selection system into one circuit, one plasmid and one strain, which allows single round within 2 days.

References

[1]1.1,1.2,1.3Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proceedings of the National Academy of Sciences of the United States of America 97, 6640-6645, doi:10.1073/pnas.120163297 (2000).

[2]Mosberg, J. A., Lajoie, M. J. & Church, G. M. Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate. Genetics 186, 791-U759, doi:10.1534/genetics.110.120782 (2010).

[3]Wang, J. P. et al. An improved recombineering approach by adding RecA to lambda red recombination. Molecular Biotechnology 32, 43-53, doi:10.1385/mb:32:1:043 (2006).

[4]Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular systems biology 2, 2006 0008, doi:10.1038/msb4100050 (2006).

[5]Sharan, S. K., Thomason, L. C., Kuznetsov, S. G. & Court, D. L. Recombineering: a homologous recombination-based method of genetic engineering. Nature protocols 4, 206-223, doi:10.1038/nprot.2008.227 (2009).