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, BAC or even multi copy plasmids, as long as each side of the donor dsDNA/ssDNA are flanked by 36-50bp homologous arms1. The homologous arms also determine precise site of recombination, thus the donor fragment can be insert into any site 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’ll see that our GeneSocket system not only utilize all the advantages of Lambda red systems but also change the whole technology to make recobineering more simple and fast.

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 recombination².

• Beta:

  Beta is a single strand binding protein, who 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’s important for using dsDNA as donor fragment.

• Homologous arm

  Homologous arm are short sequence flank over each side of the donor sequence. It also matches the recombination site on acceptor region. For high possibility of recombination, the homologous arm maybe the longer the better, but actually 36-50bp are enough for antibiotic resistance screening as most researchers has been reported1

  Table.1 Allele designations of chromosomal gene disruptions1

  the sequence of homologous arm are not limited by specific recombination site like many recombinases demand. But there are also some factors we need to consider when choosing this arm. 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 RecA3. So Lambda red system are 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. Tomoya et al., has used it to make construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection, they use the same resistance gene to recombine chromosome region rank from 20 to 7000bp4. But current strategy of using Lambda red is reliable but mainly focus on single changes, if we want to do multiple changes on chromosome as synthetic biology needed, we must try to find a new better way.

Here is one of the current strategies using Lambda red5:

With this strategy, we have at least transform two plasmids and do negative selection twice. See this page to know the different between GeneSocket method and current strategy. You’ll find who is faster and easier.

Figure.4 ZJU λ-red current method	Preparation 1.Gene of interest are going to be inserted. 2.Insertion site should be found, a series of plasmids are used to acquire resistance gene with FRT or LoxP site. 3.Fragments are prepared by PCR and ligation.
	Recombination 4.Insertion mode: GOI is link to resistance gene, two recombination sites are flanked each side. 5.Plasmid like pKD46 carry Lambda red gene to allow recombination happen.
	Resistance gene retrieve 6.pKD46 should be discard after this step, and a new plasmid like pCP20 carry recombinase are transformed into cell to retrieve resistance gene. 7.Sacr can’t be avoid by this method for the use recombinase site.

Summary

Lambda red is a broadly used recombination system, recombineering based on Lambda red also shows large protential in chromosome engineering. Generally, current recombineering strategy mainly focus on inactivate or insert single gene, so although the protocol are quite complex, it is acceptable. If we want to do continous recombination round by round, the current strategy may have trouble. So, it’s 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 allow single round within 2 days.

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References

[1]Datsenko, 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).