DMX-iGGA: 3 part lists in 3 rounds

Join 72 synthesised parts into 13,824 distinct constructs in 3 rounds, over 7 protocols. Each round opens the library with BbsI, releases one part list with BsaI, ligates the two, and transforms, grows and preps the result for the round after it.

  • blunt sitespass
  • destination openspass
  • donor releasespass
  • ends distinguishablepass
  • ligation fidelitynot judged
  • barcode spacingpass
  • barcode readingpass
  • cargo sitespass
  • cargo framepass
  • well primerspass
  • product openspass
  • barcode blockpass
  • terminal blockpass
  • terminal stoppass
  • handoffspass
  • choicespass
  • sourcespass

ligation fidelity not judged: 4 measured, including: round 1 ligation scores 1.000 on Pryor 2020 S4 Table, measured with BbsI-HF, a T4 ligase measurement cycled with the enzyme present; this round ligates with T7 DNA Ligase in StickTogether DNA Ligase Buffer

35 numbers in this run have no source: 4 bench numbers and 31 prices.

How this library is designed

One round appends one part list to every member of the library at once, so 3 rounds make 13,824 constructs out of 72 synthesised parts.

The product keeps BbsI's sites, which is what lets the next round open it, and loses BsaI's with the external stuffers.

Your vector already carried an internal stuffer, so every part enters position N on AGGA, which is the overhang that stuffer spells: DNA that exists cannot be re-chosen.

The overhang standard changes 27 amino acids across 26 part ends; changes.tsv has wild type beside synthesised, and that is what you are about to pay for.

The last round needs 195,386 colonies for a 0.99 chance that none of its 13,824 products is missing, which is 14x its products. A round short of that floor loses members no later round can put back.

The finished barcode block is 41 bp and reads in the reverse of the order the rounds ran; barcodes.tsv says which barcode names which part.

Read every well back

Both routes mark every well so that sequencing says which well a read came from, and both end with a pass or a fail for each well. Do one of them, never both.

How many wells each reaches is not what picks between them, and it does not rank them the way the bench's rule of thumb does. Barcode ligation addresses more than 330,000 wells from one plate of barcodes; index PCR addresses 9,216. Most runs sit well below either.

Barcode ligation carries a fixed cost and a fixed delay, paid once whatever the library's size: $695 of sequencing, and five days. Because they are paid once, the whole route costs less for each design the more designs there are, falling from $19.52 a design over 100 designs to $3.38 over 2,000. Nobody has published the same table for index PCR, so no price for it, and no price per well for either route, is stated anywhere here.

Index PCR runs one thermocycled reaction in every well, so thousands of wells need many thermocyclers. Barcode ligation needs none: a whole 1536-well plate marks in a single incubator. That is the only direct comparison of the two in print, and the barcode ligation route's own authors wrote it about the route they replaced; no independent measurement of one against the other was found.

Doing neither is also a choice. Every well is then enriched for the design it was built from, and nothing firmer than that can be said: the one published purity figure, 89.3% of 929 wells at 90% clonal purity or better, was measured on short arrayed fragments under 314 bases rather than on cargo from an oligo pool, and the same work found that polyclonality grows with length. Nothing measured says the material is even. To know what a well holds, read it back.

How the run fits together

    • parts to seat one part a tube, blunt and flanked so BsmBI releases it from its carrier the bench already holds it
    Part carrier
  2. Primer plates
  3. Cargo ordering and pool preparation
    • oligo pool the pool resuspended from Cargo ordering and pool preparation
    • primer working plate 1 a copy of primer stock plate, used for one run · 10 µM, 100 µL per well from Primer plates
    • block vector 1 DMX-iGGA N, ready to open the bench already holds it
    • block vector 2 DMX-iGGA DBD, ready to open the bench already holds it
    • block vector 3 DMX-iGGA C, ready to open the bench already holds it
    Cargo creation
    • cargo archive plate one well a design, polyclonal, sealed and frozen from Cargo creation
    • DMX barcode kit the lab's own barcoding plasmids, one group a picked plate the bench already holds it
    • Barcoded index primer plate the lab's own index primers, prepared once and called for by a run · 1 µM each the bench already holds it
    Read every well back do one of these two
    • part carrier plate one pCR-Blunt II-TOPO a part, one part a well, selected on KanR from Part carrier
    • clonal picked plate one well a picked colony, each well one clone from whichever way to read every well back you did
    • well calls a pass or a fail a well, and which design each well holds from whichever way to read every well back you did
    • block vector 1 DMX-iGGA N, ready to open the bench already holds it
    Library assembly in rounds
    • library prep, round 3 the pooled library after round 3, as a plasmid prep from Library assembly in rounds
    • working vector the backbone the library ends in the bench already holds it
    Final cargo ligation
  • primer stock plate 74 primers seated in a 96-well plate · 100 µM the run ends holding it
  • the library in its working vector 3 part lists joined, pooled and ready for the screen the run ends holding it

Protocols

  1. Part carrier 1 step
  2. Primer plates 5 steps
  3. Cargo ordering and pool preparation 2 steps
  4. Cargo creation 3 steps
  5. Read every well back do one of these two
  6. Library assembly in rounds 27 steps
  7. Final cargo ligation 5 steps

Schedule

ProtocolStepsHeldHolding nothing
Part carrier15 min0
No waiting recorded.
Primer plates5no sourced number5
No waiting recorded.
Cargo ordering and pool preparation2no sourced number2
No waiting recorded.
Cargo creation31 h 18 min1
No waiting recorded.
Read every well back630 min to 1 h 35 min5
Cargo validation: barcode ligation630 min5
No waiting recorded.
Cargo validation: index PCR61 h 35 min5
No waiting recorded.
Library assembly in rounds2752 h 30 min15
No waiting recorded.
Final cargo ligation515 h 45 min2
No waiting recorded.
Total4970 h 8 min to 71 h 12 min over 5 of 7 protocols30

A blank here is a number nobody has stated, never a zero. Most of this run is time nobody attends and nobody can date, so it is written down rather than drawn as a length. No protocol here states hands-on or unattended time, so those columns are left out. Where one job offers several ways, the row above them gives each column's range: you do one way, and that way's own row says what it takes. A total that covers only part of the run says so beside it.

Holes

35 numbers this run would otherwise have to invent. None is filled with a guess, and each says below what it waits on: a bench, a shelf, a price, or a number nobody has published.

  • P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, P20, P21, P22, P23, P24, P25, P26, P27, P28, P29, P30, P31, P32, P33, P34, P35, P36, P37, P38, P39, P40 no sourced number — 31 numbers, each waiting on the same thing. Waiting on a price record. Filled by a quantity the design sizes, and a price record holding a row for this item. Each stands on Reagents and equipment.
    • P10 no sourced number — pCR-Blunt II-TOPO, money: nothing prices this item.
    • P11 no sourced number — Zero Blunt TOPO PCR Cloning Kit, money: nothing prices this item.
    • P12 no sourced number — nuclease-free water, money: nothing prices this item.
    • P13 no sourced number — Empty 96-well plate, money: nothing prices this item.
    • P14 no sourced number — Adhesive plate seals, money: nothing prices this item.
    • P15 no sourced number — NEBridge Golden Gate Assembly Kit, BsmBI-v2, money: nothing prices this item.
    • P16 no sourced number — Q5 DNA Polymerase, money: nothing prices this item.
    • P17 no sourced number — dNTP mix, money: nothing prices this item.
    • P18 no sourced number — DMX-iGGA vector, money: nothing prices this item.
    • P19 no sourced number — CutSmart Buffer, money: nothing prices this item.
    • P20 no sourced number — SPRI paramagnetic beads, money: nothing prices this item.
    • P21 no sourced number — Recovery medium, money: nothing prices this item.
    • P22 no sourced number — Selective broth and plates, money: nothing prices this item.
    • P23 no sourced number — 25 cm BioAssay plate, money: nothing prices this item.
    • P24 no sourced number — 384-well culture plate, money: nothing prices this item.
    • P25 no sourced number — 1536-well barcoding plate, money: nothing prices this item.
    • P26 no sourced number — DMX barcode kit, money: nothing prices this item.
    • P27 no sourced number — Barcoding master mix, money: nothing prices this item.
    • P28 no sourced number — 96-well half-skirted PCR plate, money: nothing prices this item.
    • P29 no sourced number — Barcoded index primer plate, money: nothing prices this item.
    • P30 no sourced number — ThermoPol Reaction Buffer, money: nothing prices this item.
    • P31 no sourced number — Taq DNA Polymerase, money: nothing prices this item.
    • P32 no sourced number — DMSO, molecular biology grade, money: nothing prices this item.
    • P33 no sourced number — N part list, money: nothing prices this item.
    • P34 no sourced number — DBD part list, money: nothing prices this item.
    • P35 no sourced number — C part list, money: nothing prices this item.
    • P36 no sourced number — T7 DNA Ligase, money: nothing prices this item.
    • P37 no sourced number — StickTogether DNA Ligase Buffer, money: nothing prices this item.
    • P38 no sourced number — pLVX-TetOne-dom vector, money: nothing prices this item.
    • P39 no sourced number — PaqCI, money: nothing prices this item.
    • P40 no sourced number — Selective plates for the final transfer, money: nothing prices this item.
  • H29 no sourced number — PCR1, the polymerase the cycle count is stated against: no cycle count published for Q5 on a Twist oligo pool. Waiting on a number nobody has published. Filled by a count Twist states against Q5, or a pilot on this pool. Stands on Cargo creation.
  • H30 no sourced number — PCR2, the cycle count: no cycle count for PCR2, which pulls one block out of an already-amplified batch. Waiting on a number nobody has published. Filled by a pilot titrated against the heteroduplex hump on capillary electrophoresis, or a real-time run stopped before the curve plateaus. Stands on Cargo creation.
  • IDX1 no sourced number — index PCR, the pair marking one well: no index mark set is named for the barcoded primer pairs. Waiting on the lab's own stock. Filled by the prepared primer plate the lab holds. Stands on Cargo validation: index PCR.
  • H23 no sourced number — the split digest, the procedure: nobody has published two enzymes run in one tube, the second added to the first's reaction with no heat kill before the beads; the one source is the paper itself. Waiting on a number nobody has published. Filled by nothing; it is the method's own. Stands on Library assembly in rounds.