Integrating plasmid Design
Selecting an integrating plasmid
See worflows under the Protocols page. Users should plan their ORBIT experiment according to what final locus structure they want to obtain. If a user wants to keep the entire integrating plasmid in the locus, with the antibiotic resistance marker and origin, or they want to use FLP excision, then any of the original integrating plasmids (e.g. pInt_attP1_kanR, pInt_attP1_chlorR) should suffice. If users want clean deletions, then pInt_attP1_sacB_kanR should be used in conjunction with a second oligo recombineering step with the clean deletion oligo lacking attB. If users want an attB scar (38bp), then pInt_attP1_tsXis_sacB_kanR should be used.
Getting plasmids from addgene
Nearly all plasmids used in the NAR paper are available from addgene. We strongly recommend you request plasmids through this nonprofit, because they are a fantastic community resource that has standardized and simplified the MTA process. Please contact the Saunders Lab if there is a plasmid that you require that is not on Addgene, or if the cost is prohibitive for you.Â
The set of plasmids that you should request from addgene will depend on your primary use cases for ORBIT. At a minimum, users should request a helper plasmid and an integrating plasmid. We would recommend pHelper_TS_V2_ampR and pInt_attP1_kanR as the simplest set to use and request. However, if you are planning to clone constructs onto the integrating plasmid you may like the cloning site on pInt_attP1_LCS_kanR. Perhaps you should go ahead and get different antibiotic markers to ensure you have compatible options (e.g. pInt_attP1_chlorR.
The other major thing to consider is if you will be constructing clean deletions or markerless mutations (38 bp attB scar). In those cases you must request pInt_attP1_sacB_kanR and pInt_attP1_tsXis_sacB_kanR respectively.
The orthogonal att sites can be useful, and we have tried to provide a convenient set on addgene. However, it is also trivial to introduce the attP mutations into any attP1 plasmid, so if you are trying to request fewer plasmids we recommend you just start with attP1. Likewise, the various antibiotic markers can be easily swapped between plasmids. We typically use gibson type cloning - the only trick is that integrating plasmids (i.e. pInt) must be cloned into a pir+ host strain, which can replicate it (e.g. BW25141).
Integration locus structure
The structure of the ORBIT modified locus will depend on the targeting oligo and integrating plasmid. The most critical factor to consider at this stage is the attB direction. In the targeting oligo app, the attB direction is input as '+' or '-' relative to the genome. The figure below shows a '+' attB targeting oligo that deleted hisA (a fwd strand gene), and then has pInt_attP1_kanR integrated. Therefore the forward integration of the integrating plasmid results in the antibiotic marker and origin pointing to the right. An attB '-' would point the integrating plasmid in the opposite direction. Note that this gets confusing when ordering the targeting oligo, because the attB sequence in the oligo is also dependent on whether the oligo is the fwd or rev strand sequence. The targeting oligo app figures this out automatically. See further explanation under Oligo design.
We strongly recommend creating annotated sequence files (e.g. in Benchling / Snapgene) with the entire expected locus. This makes designing colony PCR primers and aligning sanger sequencing files much easier, which is essential for verifying strains. Generally, we recommend testing for the genome:pInt junctions by colony PCR as an initial test. The more stringest test would be to perform a "spanning PCR" that amplifies the whole modified locus using upstream and downstream genomic primers.
