ORBIT for E. coli : Kilobase-scale oligonucleotide recombineering at high throughput and high efficiency
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Abstract
Microbiology and synthetic biology depend on reverse genetic approaches to manipulate bacterial genomes; however, existing methods require molecular biology to generate genomic homology, suffer from low efficiency, and are not easily scaled to high throughput applications. To overcome these limitations, we developed a system for creating kilobase-scale genomic modifications that uses DNA oligonucleotides to direct the integration of a non-replicating plasmid. This method, Oligonucleotide Recombineering followed by Bxb-1 Integrase Targeting (ORBIT) was pioneered in Mycobacteria , and here we adapt and expand it for E. coli . Our redesigned plasmid toolkit achieved nearly 1000x higher efficiency than λ Red recombination and enabled precise, stable knockouts ( < 134 kb) and integrations ( < 11 kb) of various sizes. Additionally, we constructed multi-mutants (double and triple) in a single transformation, using orthogonal attachment sites. At high throughput, we used pools of targeting oligonucleotides to knock out nearly all known transcription factor and small RNA genes, yielding accurate, genome-wide, single mutant libraries. By counting genomic barcodes, we also show ORBIT libraries can scale to thousands of unique members (>30k). This work demonstrates that ORBIT for E. coli is a flexible reverse genetic system that facilitates rapid construction of complex strains and readily scales to create sophisticated mutant libraries.
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I don't believe this is a problem here, because the Bxb-1 integrase is on the helper plasmid and the integrase recognition site (attP) is on the integrating plasmid. So, during cloning, the integrase and attP site should never interact. Also, the Bxb-1 integrase is under the arabinose inducible system, although there is clearly leaky expression.
All that said, the low yield we get from the integrating plasmids is probably just due to their origin of replication. As I say in the preceding sentence, we actually tried a strain with the "pir116" allele that is supposed to replicate these types of plasmids at higher copy number, however, we observed horrific plasmid concatemers (thanks to nanopore sequencing).
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Yes, I agree this is an interesting question and I do not believe it is totally understood how large deletions are made. This paper (Maresca et al. 2010) has the most detailed information / model that I could find, based on work with the lambda Red Beta protein (https://doi.org/10.1186/1471-2199-11-54). I will try to include a comment about this when I revise the paper.
Discussion: "...the homology arm at the 3' end of the [lagging strand oligo] should anneal before the homology arm at the 5' end of the [lagging strand oligo] because its' complementary sequence is exposed earlier at the replication fork. Hence Redβ likely stabilizes annealing of the 3' homology arm while the replication fork continues. Later, when the region complementary to the 5' homology arm is exposed, the 5' homology arm anneals and the heteroduplex that promotes …
Yes, I agree this is an interesting question and I do not believe it is totally understood how large deletions are made. This paper (Maresca et al. 2010) has the most detailed information / model that I could find, based on work with the lambda Red Beta protein (https://doi.org/10.1186/1471-2199-11-54). I will try to include a comment about this when I revise the paper.
Discussion: "...the homology arm at the 3' end of the [lagging strand oligo] should anneal before the homology arm at the 5' end of the [lagging strand oligo] because its' complementary sequence is exposed earlier at the replication fork. Hence Redβ likely stabilizes annealing of the 3' homology arm while the replication fork continues. Later, when the region complementary to the 5' homology arm is exposed, the 5' homology arm anneals and the heteroduplex that promotes recombination is established."
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Hello Jase, thank you for your comments. I sincerely appreciate it and I hope this type of feedback becomes more routine in science!
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Past work suggested that smaller genomic modifications would be more efficient than largergenomic modifications (21).
It's not obvious to me how a deletion gets made by oligo recombineering. Figure 1a seems to show a single oligo binding both upstream and downstream of the region to be deleted, but is this what happens with very large deletions? More than likely, individual recombineering events are taking place at two sites, then the intervening sequence is removed with some frequency. It would be helpful to have more information or data regarding the suspected mechanism of large genomic deletions
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Consequently, we believe that pHelper_V2 will be a usefulORBIT tool moving forward to avoid elevated background mutation rates. However, for theremainder of this work, we continued to employ the V1 helper plasmid, since all previous workwas with this plasmid.
Again, thank you to the authors for providing a clear explanation of their work and their interpretations of the results.
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Past work also recommended using phosphoorothioate (PO) bonds for recombineering oligos tomake the single stranded DNA resistant to endogenous exonucleases (21). For oligos targetingthe galK locus, we found only minor increases in efficiency by adding PO bonds (Fig S2A).Phosphoramidite chemistry-based DNA oligo synthesis is known to yield larger fractions ofincomplete products as oligo lengths increase, therefore DNA synthesis companies oftenrecommend paying extra for high quality purifications to obtain more full-length sequences. Wetested different oligo purifications (desalting, cartridge, HPLC, PAGE) for the galK deletionoligo and found that PAGE purification did increase efficiency (~2.5x), but purification was notnecessary for most applications (Fig S2B). We also tested 90 nt targeting oligos for all 4 locifrom 3 different …
Past work also recommended using phosphoorothioate (PO) bonds for recombineering oligos tomake the single stranded DNA resistant to endogenous exonucleases (21). For oligos targetingthe galK locus, we found only minor increases in efficiency by adding PO bonds (Fig S2A).Phosphoramidite chemistry-based DNA oligo synthesis is known to yield larger fractions ofincomplete products as oligo lengths increase, therefore DNA synthesis companies oftenrecommend paying extra for high quality purifications to obtain more full-length sequences. Wetested different oligo purifications (desalting, cartridge, HPLC, PAGE) for the galK deletionoligo and found that PAGE purification did increase efficiency (~2.5x), but purification was notnecessary for most applications (Fig S2B). We also tested 90 nt targeting oligos for all 4 locifrom 3 different suppliers with different price points ($7-$18 per oligo) and found obviousdifferences, suggesting variable oligo quality (Fig S2C). Typically, we used 1 μM final oftargeting oligo (assuming 50 μL aliquots), but we found that efficiency does not increasemonotonically with increasing oligo concentration. Instead, we found that 10 nM oligo supportsefficiency above background, and 100 nM oligo was consistently the most efficientconcentration (Fig. 1E)
This experiment, in which the authors detail the effects of common variables, is so useful to potential users and so often left out of publications. I praise the authors for reporting the results here
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Materials and Methods:
I have not attempted to reproduce the protocols presented, but the authors provide clear, extensive, and detailed instructions for all steps of the method and their experiments. The methods section is exemplary.
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To simplify this process, we developed a web app that instantaneously generates targetingoligos that bind the lagging strand and face the same direction as a target gene
I tried this little editor. It is not only very functional, but also fast and intuitive. Great job and much appreciated from a user perspective.
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Often, low yield integrating plasmids were midior maxiprepped (Zymo Research #4200 / #4202) using the low copy number protocols, whilehigher yield replicating plasmids were miniprepped.
integrases can lead to lots of genetic instability. i wonder if a repressible system would help to clone these constructs when expression is not needed.
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This method, Oligonucleotide Recombineering followed by Bxb-1 Integrase Targeting(ORBIT) was pioneered in Mycobacteria, and here we adapt and expand it for E. coli. Ourredesigned plasmid toolkit achieved nearly 1000x higher efficiency than λ Red recombinationand enabled precise, stable knockouts (<134 kb) and integrations (<11 kb) of various sizes.Additionally, we constructed multi-mutants (double and triple) in a single transformation, usingorthogonal attachment sites. At high throughput, we used pools of targeting oligonucleotides toknock out nearly all known transcription factor and small RNA genes, yielding accurate,genome-wide, single mutant libraries.
The authors apply the ORBIT genome engineering method in E. coli. The method, providing large and scalable insertions and deletions in the genome, involves targeted integration of …
This method, Oligonucleotide Recombineering followed by Bxb-1 Integrase Targeting(ORBIT) was pioneered in Mycobacteria, and here we adapt and expand it for E. coli. Ourredesigned plasmid toolkit achieved nearly 1000x higher efficiency than λ Red recombinationand enabled precise, stable knockouts (<134 kb) and integrations (<11 kb) of various sizes.Additionally, we constructed multi-mutants (double and triple) in a single transformation, usingorthogonal attachment sites. At high throughput, we used pools of targeting oligonucleotides toknock out nearly all known transcription factor and small RNA genes, yielding accurate,genome-wide, single mutant libraries.
The authors apply the ORBIT genome engineering method in E. coli. The method, providing large and scalable insertions and deletions in the genome, involves targeted integration of an integrase target site (AttP) by oligo recombination, followed by integration of a larger construct at the newly generated AttP locus. Overall, the method combines the ease of oligo recombineering with gene- and pathway-scale genomic changes that are needed to test large synthetic libraries. This work applies ORBIT to E. coli, shows impressive performance, and opens the door for future screening with this system.
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