Big-IN Payloads

See our resources page for plasmids available from Addgene

BIN-IN Payload (aka Assemblons) vectors are BAC-YAC constructs that facilitate yeast cloning and bacterial copy number induction before delivering payloads to Big-IN Landing Pads (LPs). We are currently using almost exclusively pLM1110 as our Payload Assembly Vector.

pLM1110 contains the following elements/regions:

  • Yeast parts:

    • CEN/ARS. Note: the CEN/ARS region contains a 778 bp region that was carried over inadvertently from the E.coli genome and seems to be required now (efforts to remove it failed).

    • LEU2 selection marker

  • Bacterial parts:

    • KanR selection marker

    • OriV and Ori2

    • SopA/B/C: these are required for the copy number induction in bacteria

  • Mammalian parts:

    • The vector contains a backbone selection/sorting cassette (pEF1a-GFP-T2A-BSD). We use this feature to transiently select for cells that were successfully transfected with the payload DNA.

  • Cloning parts:

    • I-SceI sites: a pair of I-SceI sites allow replacement of the RFP region with the Payload/Assemblon. Note that the LoxM/LoxP sequences are lost upon I-SceI digestion.

    • Lvec/Rvec: these two regions serve as universal linkers for yeast assembly of vector and payload.

    • BsaI sites (external to the lox sites) enable Golde Gate Assembly.

There are other types of Big-IN Payload Vectors:

  • pLM1050: Similar to pLM1110, but lacking the backbone pEF1a-GFP-T2A-BSD selection/sorting cassette.

  • pLM1081: Similar to pLM1110, but lacking the backbone pEF1a-GFP-T2A-BSD selection/sorting cassette and with a URA3 yeast selection marker instead of LEU2.

  • pPL001: Similar to pLM1110, but the pEF1a-GFP-T2A-BSD selection cassette is inside the lox sites. This allows positive selection of the Assemblon/Payload in mammalian cells, resulting in a non-scarless delivery. Note that I-SceI digestion would remove the selection cassette, but it can be reassembled with the Assemblon/Payload. This plasmid can also serve as a small payload for positive control delivery to LPs.

  • pPL001-BBTK: Similar to pPL001, but also containing a backbone negative selection cassette (phPGK-ΔTK-SV40pA). Allows GCV counterselection of backbone/off-target payload integration.

  • pPL001FF-BBTK: Similar to pPL001-BBTK, but also contain two FRT sites flanking the pEF1a-GFP-T2A-BSD cassette, which theoretically (never tested), allow removal of the scar.

Delivery of Payloads to Mammalian Cells:

We are routinely delivering large (~150 kb) payloads to mESCs. For delivery to mESCs we transfect, using the Amaxa nucleofector 2b, a total of ~10 µg payload DNA (prepped with the Nucleobond Xtra BAC kit and eluted so that the concentration is ideally >1 µg/µl). Together with the payload DNA, 2-5 µg of pCAG-iCre is co-transfected. Typically we transfect 5M cells and plate them in one 10cm dish. If the payload is small (e.g. <100 kb), consider also plating a sparser condition as the dish can reach density too soon (before Proaerolysin counterselection can be safely applied). 

The cell selection scheme, for scarless delivery to PIGA-harboring LPs, is as follows: On d1 post-transfection, select with 10 µg/ml Blasticidin-S for 2 days (with one media change on d2 post-transfection). This will select for rare (typically <1%) cells that were transfected with the payload DNA (if pLM1110 is the backbone). On d3, remove selection and maintain cells for an additional 4 days with media change when needed. On d7 (or d6 if cells are too dense), apply 2 nM Proaerolysin for 2 days (with one media change). This should eliminate the vast majority of colonies. Maintain cells for an additional 1-4 days before picking clones.

Variations on this selection scheme include:

  • When delivering non-scarless payloads, maintain BSD selection throughout the process.

  • When delivering payloads with a backbone TK counterselection cassette (BBTK), select with GCV for 2-3 days after selecting with Proaerolysin (e.g. on days 9-11 post-transfection).

If it's your first time doing Big-IN delveries, we highly recommend including positive control payloads. There are a few options:

  1. pPL001 (aka pPL1). This payload contains a loxM-pEF1a-GFP-T2A-BSD-loxP cassette (2.7 kb lox-to-lox), so you can positivey select for its integration by keeping BSD selection on beyond day 2 post-tranfection and later on apply negative selection. If your cells do not survive this, something wrong with your LP or transfection. The vast majority (if not all) pos/neg-selection-surviving colonies should be GFP positive and correct.

  2. pPL001-BBTK. Similar to pPL001 but also have a backbone TK cassette for negative selection (which isn't a real issue with Big-IN).

  3. Coming soon - more positive control payloads on Addgene.

Quality Control:

After delivery, clones are screened and verified for correct integration using PCR. This is usually performed on crude gDNAs extracted from cells grown in 96W plates using the Quick and Dirty gDNA Prep. PCR genotyping is similar to the process of verifying correct LP clones. In essence, we screen for:

  • The presence of the two newly-formed junctions between the delivered payload and its surrounding genome sequence. This is tricky when performing allelic Big-IN and delivering a payload that restores the wild-type (WT) junctions. The problem is that those junctions are preexisting in the second unedited allele and hence hard to screen for. One solution is using hybrid mESCs, which allow screening with allele-specific primers. Otherwise, unique primer binding sites can be added at the edges of the payload to generate unique amplicons.

  • The presence of one or more payload-internal regions. This is useful for identifying and eliminating clones in which there are deletions in the payload. Similarly to the junction assays, here too allele-specific assays are useful, but often other options exist. If the payload contains any unique sequence (or deletion), those can be screened for.

  • The absence of payload backbone integration. This is rare, but can happen, and can be screened with primers that target the payload Ori (which is different than the LP Ori), BSD etc.

  • The loss of the Landing Pad. In rare cases, the LP is silenced and thus cells can survive negative selection with Proaerolysin without losing the LP. Screening for LP presence can identify such LP-silenced clones.

  • Note: we now routinely perform payload integration screening using a 384W qPCR, which enables screening 96 clones for 4 different assays. To this end, the genotyping primers should be "qPCR-grade" (short amplicons). Another trick is to include in the same well more than one negative assays, e.g. to include both Ori and BSD primers (or Ori and LP primers). Using the ECHO to load plates saves time (contact Raquel Ordonez for protocols).

Clones identified during the initial screening are expanded (usually from 96W to 24W and then to 6W plates). High quality gDNA is prepared using the Qiagen kit and submitted to Capture-seq with bait that includes a BAC (or BACs) covering the engineered region, the pCAG-iCre plasmid (to verify it did not randomly integrate), the LP plasmid (to verify its loss) and usually pPL001 or another payload vector to capture the payload backbone.