ORBIT: Protocols

Workflow overview

ORBIT is a complete reverse genetic toolkit, therefore the types of modifications that can be made vary and should be taken into consideration when planning the initial ORBIT experiment. The flowchart below provides an overview of how to approach an ORBIT experiment depending on whether the strain you are making will retain the entire integrating plasmid or have part (or all) of it removed (markerless / scarless modification).

Before starting, you should select the appropriate helper plasmid, integrating plasmid and targeting oligo. 

The helper plasmids generally function equivalently - there are only minor differences like antibiotic markers and temperature sensitive vs. standard origins. Note: we do generally recommend using V2 helper plasmids to avoid off target mutations that accumulate with V1 plasmids.

The integrating plasmids will vary significantly depending on the desired final locus. All integrating plasmids are suitable for FLP/FRT excision of the plasmid backbone and antibiotic marker. Clean deletions require the pInt_attP1_sacB_kanR plasmid. Markerless mutations with the attB scar require pInt_attP1_tsXis_sacB_kanR. Users may clone various constructs onto the integrating plasmid, and of course those integrating plasmids should be used for those purposes. Users should ensure that antibiotic markers are not duplicated (i.e. do not use pInt_attP1_ampR with pHelper_TS_V2_ampR). Finally, if orthogonal att sites are being used (i.e. not the standard WT attB1/attP1), care should be taken to pair matching att sites on the targeting oligo and integrating plasmid (e.g. TO with attB2 and pInt_attP2_kanR). See further details on the Integrating plasmid page.

The targeting oligo should be designed and ordered appropriately. For example, an oligo to delete a gene should be designed differently than an oligo to create an intergenic insertion. See further details on the Oligo design page.

Key Steps

As explained in the workflows section, there are only a few details that are important for each step. These steps can be logically mixed and matched to achieve the desired results.

Below are some quick, plain text protocols. More detailed pdf protocols can be found below.

RecT induction + electrocompetent cell prep

This protocol to induce oligo recombineering should be used anytime an oligo is going to be introduced to cells (targeting oligos or clean deletion oligos).

Electroporation & Recovery

This protocol can be used anytime an ORBIT transformation is performed, or when transforming just an oligo (e.g. clean deletion) or just a plasmid (e.g. pHelper or pCP20 for FLP). 

Helper plasmid curing

Protocol PDFs

The detailed protocols below walk through how to make induced electrocompetent cells and perform ORBIT integrations. Combining these protocols with the overview above should enable many avenues of strain construction.