ORBIT: Introduction

Concept

ORBIT was originally developed for use in Mycobacteria by Kenan Murphy in a landmark paper and it stands for Oligo Recombineering followed by Bxb-1 Integrase Targeting. The Saunders Lab has modified and improved this system for use in E. coli.

Single stranded DNA (oligo) recombineering has been used for E. coli genetics for almost 20 years (See Donald Court). The central idea is that oligos that contain homology to the genome are bound by a single stranded DNA annealing protein (e.g. Lambda Red Beta), which binds them to a single stranded genomic target during DNA replication. These fragments are thought to act like Okazaki fragments on the lagging strand that are then directly incorporated into genomic strands.

Small deletions (and insertions) can be made using these oligos, because the homology arms can bind two distinct positions and cause the target strand to loop out. The genomic strands eventually segregate into distinct double stranded genomes and therefore mutants can be identified that have the modification of interest.

The primary issue with this flavor of genetics, is that unlike traditional reverse genetic methods, there is no antibiotic resistance that can be used to select for recombinants. Oligo based methods are quite efficient, so small mutations can be identified simply by screening colonies (e.g. 10% efficiency means screening only 10 colonies), but this doesn't easily work for larger mutations that occur at lower frequencies (e.g. 0.1% efficiency means screening 1000 colonies). However, if there was a way to incorporate antibiotic selection, then mutations of any size could be specified by an oligo.

ORBIT solves this problem by using a targeting oligo that not only contains homology arms, but also a site called attB. This is an attachment site that will recombine with the corresponding attP site in the presence of the integrase, Bxb-1. By putting attP on a non-replicating plasmid, Bxb-1 will insert the plasmid into the newly formed attB locus specified by the targeting oligo. This effectively means that one can specify a mutation with an oligo, but select for recombinants using the antibiotic resistance from the plasmid. The amazing part about this two step process, is that it can be completed in a single co-electroporation with the oligo and the plasmid, making it very convenient.

Check out our Nucleic Acids Research paper for much more detail!

Execution

To perform ORBIT you basically need three things:

1. Strain of interest with helper plasmid

2. Targeting oligo

3. Integrating plasmid

Once you have these components, you need to 1) induce the oligo recombineering genes on the helper plasmid and prepare competent cells 2) co-electroporate the targeting oligo and integrating plasmid 3) recover and induce Bxb-1 and 4) plate for recombinants. The protocols page has more details, but if it's successful you should be able to rapidly and efficiently make mutants!

Helper plasmid

The ORBIT helper plasmid has two separate inducible modules that perform the two conceptual steps of ORBIT.

First, there is the oligo recombineering module, that includes a single stranded DNA annealing protein (CspRecT). This highly efficient oligo recombineering system was discovered and characterized by Wannier et al. 2020. In version 1 plasmids, there is a mismatch repair suppression protein (dominant negative mutation of MutL - E32K), however, this caused elevated levels of background mutations and was removed in version 2 helper plasmids. Generally, we strongly recommend V2 plasmids, however, it is possible that V1 plasmids are slightly more efficient and they should be much better at incorporating point mutations encoded in oligos.

This oligo recombineering module is induced with xylS, which responds to m-toluic acid. This inducer is cheap and easy to order from Sigma Aldrich (#T36609) and is soluble in 100% ethanol.

Second, there is the Bxb-1 integrase that catalyzes the integration of the non-replicating plasmid into the attB site. This gene is simply arabinose induced.

Once ORBIT is complete, users should remove the helper plasmid before using their recombinant strain, so helper plasmids either contain sacB (for sucrose counter selection) or temperature sensitive origins. We highly recommend the pHelper_TS_V2_ampR plasmid!

Fully annotated genbank files are available here.

Targeting oligo

The targeting oligo has three parts - the upstream homology arm, the downstream homology arm, and the attB site in between. The attB site is 38 bp, and the minimum recommended homology length is 52 bp, which gives a 90 nucleotide (nt) oligo or longer. These oligos can ordered directly from IDT or Millipore Sigma and do not require any modifications or special purifications beyond desalting. These oligos can essentially be diluted in water and used as-is, making it very convenient.

For our lab, oligos 90, 100, or 120 nt are available from IDT and cost $16, $42, or $82 respectively. Oligos 90 - 120 nt are available from Millipore Sigma and cost $18-24 (UTSW 2023 pricing). In general, the longer the homology arms, the more efficient ORBIT will be, so we typically use 120 nt oligos from Millipore Sigma, although IDT oligos of the same length (i.e. both 90 nt) tend to be a bit more efficient.

The targeting oligo should target the lagging strand template, and when taking this into account and the possible +/- direction of the attB site things quickly become complicated. Therefore we wrote a simple app that will give you a targeting oligo taking these things into account for a specified gene or genomic location. You can run this app online and the code is also open source, which allows anyone to run the app locally or modify it for their own purposes.

Integrating plasmid

The final piece of ORBIT is the non-replicating or integrating plasmid. This plasmid contains the attP site (48 bp) that Bxb-1 recognizes and integrates into the new genomic attB site. For deletions, it doesn't really matter what is on this plasmid, because most of the plasmid backbone can be excised using the classic FLP recombinase (e.g. pCP20).

However, this integrating plasmid can also be used to insert constructs onto the genome. This is particularly useful for transcriptional reporters, so there is a derivative of the integrating plasmids that contains a library cloning site flanked by transcriptional terminators. In this case, the entire plasmid backbone, except for the desired construct can be excised with FLP.

Normal molecule cloning works with this plasmid as long as it is propagated in a pir+ host strain.

Markerless / scarless strategies

Various strategies can be taken to remove part or all of the integrating plasmid backbone - leaving markerless or scarless modifications. There are three options presented in the manuscript. A protocol overview is provided on the Protocols page.