EarlyBac

EarlyBac manual

	pFp10*	pUCDMp10	pSPLp10
One protein*	√
Two proteins**	√	√
Two proteins**	√		√
Three proteins	√	√	√

*Always subclone your first gene into pFp10. If you are expressing one protein, subclone into pFp10

**Subclone your 2^nd gene into pUCDMp10 or pSPLp10

Amplification of plasmid vectors

pFp10 – use DH5alpha – Ampicillin resistance

pUCDMp10* – use PIR1 from Thermofisher (cat#: C101010) – Chloramphenicol resistance

pSPLp10* – use PIR1 from Thermofisher (cat#: C101010) – Spectinomycin resistance

*Low copy

The above vectors were made in the backbones of pFL, pUCDM, and pSPL multibac vectors ( Fitzgerald et al Nat Methods. 2006 Dec;3(12):1021-32. doi: 10.1038/nmeth983.)

Cre-lox recombination (for multiple protein expression)

-We typically recombine two vectors simultaneously (three may be done with low efficiency).

First round

pFp10 + pUCDMp10 = pFp10/pUCDMp10

pFp10 + pSPLp10 = pFp10/pSPLp10

Second round

pFp10/pUCDMp10 + pSPLp10 = pFp10/pUCDMp10/pSPLp10

pFp10/pSPLp10 + pUCDMp10 = pFp10/pSPLp10/pUCDMp10

-We make our own Cre-recombinase, but it can also be purchased from NEB (https://www.neb.com/products/m0298-cre-recombinase#Product%20Information)

Quick expression test by transient transfection

-Quick expression experiments (e.g., FSEC) can be done by transfecting the vectors into insect cells before making virus.

-We transfect into HighFive cells for FSEC since the transfection efficiency is higher than Sf9 cells. Culture HighFive cells in ESF921.

-Use Cellfectin II (Thermofisher) as a transfection reagent.

Bacmids

-We happen to use DH10EmBacY (Geneva-Biotech, https://geneva-biotech.com/product_category/insect-cell-expression/multibac/). This bacmid expresses YFP, which serves as an infection marker. The viral genes v-cath and chiA are also disrupted.

-DH10Bac (Thermofisher, https://www.thermofisher.com/order/catalog/product/10361012?us&en#/10361012?us&en) can also be used.

-Transposition can be done using the methods described in Thermofisher or Geneva technology (https://geneva-biotech.com/wp-content/uploads/2019/12/MultiBac-Manual-v8.4.pdf)

Transfection/Virus amplification

-Sf9 cells (e.g., six-well plates containing 3X10^6 cells in 2 ml of media per well)

-We use TransIT-2020 or Fugene for transfection.

-Wait 4-5 days after transfection and collect supernatant (P1 virus).

-We like to do transfection and virus amplification in Hyclone CCM3 medium.

Mid-large scale expression

-We culture Sf9 cells in Hyclone CCM3 medium. We have not done an extensive media screening but see a higher expression level in CCM3 than in Sf900-III when using EarlyBac.

-For membrane protein expression, we grow Sf9 cells to 4X10^6 cells/ml (0.8-1 L in 2 L flask; 110 rpm at 27^oC).

-For secretion expression, we grow HighFive cells to 1.5-2X10^6 cells/ml (0.8-1 L in 2 L flask; 110 rpm at 27^oC). Use ESF921 medium for culturing HighFive cells.

By default, we collect cells 48-h post-infection. Remember, you are using an early promoter, not a late promoter; thus, the protein expression starts immediately after infection (~12 h).