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.

• RecT induction + comp cell prep (fresh or frozen, same for original mod or clean deletion)

• Electroporation (same for original mod, multi TO, or clean deletion)

• Recovery

• Curing

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).

1. Inoculate strain + pHelper into fresh media + helper antibiotic (typically from overnight culture).

2. Grow to OD ~0.3.

3. Add 1mM m-toluic acid (typically from 1000x stock of 1M m-toluic acid in ethanol) and return to incubator for 30 min induction.

4. Place cultures on ice for at least 15 min (swirling occasionally).

5. Wash 3x with ice cold water (or 10% glycerol if making frozen aliquots). Typically 50 mL conicals are used for large volumes with 5-10 min spins at 5,000 x g. However, smaller volumes can be washed in 1.7 mL microfuge tubes (1 min spin at 12,000 x g) - we commonly do this for the second transformation step of clean deletion.

6. Aliquot accordingly (typically 50 µL). We commonly set up empty tubes on ice. For storing aliquots at -80°C, we recommend transferring to dry ice for 10 min then transferring to the ultracold freezer.

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). 

1. Pre chill electroporation cuvettes on ice for at least 10 min.

2. Thaw frozen electrocompetent cell aliquots for 5 min on ice (or use fresh aliquots).

3. Add integrating plasmid (typically 100 ng) and targeting oligo (typically 2 µL of 25 µM stock) to cell aliquots. If the transformation is not for ORBIT, add the appropriate oligo or plasmid here.

4. Electroporate with standard E. coli settings (1.8 kV, 25 μF, 200 Ω for 1 mm cuvettes) and immediately add 1 mL of recovery media.

5. Transfer cells + recovery media to culture tube. For ORBIT, this recovery should be 3 mL of LB + 0.1% arabinose. For other transformations, arabinose is not required.

6. Let recover shaking for at least 1 hr at 37°C (standard), or 30°C if a temperature sensitive plasmid needs to be maintained.

7. Plate on a selective antibiotic. Typically this will be the antibiotic marker on the integrating plasmid. The easiest way to plate is to spin down the culture and concentrate in <200 uL of volume. Then streak that liquid for isolation.

Helper plasmid curing

• The temperature sensitive helper plasmid can be cured by recovering and plating at 37-42°C. Steps 6 & 7 above.

• The standard helper plasmids (i.e. pHelper_V2_gentR) can be cured with sucrose counter selection. Grow a colony in liquid (LB + integrating plasmid antibiotic) to saturation, then streak plate 100 µL onto a sucrose plate (7.5% sucrose in standard LB). Alternatively, the ORBIT recovery from step 6 above can be diluted 1:100 into liquid LB + integrating plasmid antibiotic and allowed to grow overnight. The next day cells can be plated onto sucrose.

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.