Plasmid Production is a Three Step Process

Plasmid Production involves three main steps: DNA Synthesis, DNA Assembly, and DNA Sequencing. In this context, "DNA synthesis" refers to printing DNA — chemically or enzymatically stringing together single base pairs outside of a cell. "DNA assembly" refers to molecular cloning or stitching larger double-stranded pieces of DNA into a circular plasmid. “DNA sequencing” means reading the assembled plasmid to verify that it is correct. 

Right now, these three steps are typically done in three separate places. Synthesis is outsourced to Twist, Ansa, IDT, Elegen, or Genscript. Assembly is done at the scientist’s lab, then shipped or dropped off at a sequencing company like Plasmidsaurus or GeneWiz. 

From a molecular biologists perspective, the whole process looks like this— don’t worry too much about the technical details here they are only included to explain the timeline:

1. Design your plasmid and cloning strategy (day 1)

2. Order the parts (day 1)

3. Synthesis company produces fragments

4. Once DNA parts are received, PCR amplify them (day 7)

5. Restriction Enzyme Digests and Ligation (day 7)

6. Transform into E.coli (day 7)

7. Plate on selective media, grow overnight (day 7)

8. Pick colonies into liquid culture, grow overnight (day 8)

9. Miniprep (harvest the plasmids from the cells) (day 9)

10. Send purified plasmid to be sequenced (day 9) 

11. Analyze sequencing results (day 11)

There is leniency in these timelines because, for now, science is executed by humans. There will always be that unexpected meeting which delays the transformation, the parts get delivered late, the DNA concentration is too low to be useful so you must re-miniprep it, you miss the FedEx dropoff time, or you bump into the weekend (and have boundaries) — there are many opportunities for delay. 

There are also many reasons not caused by human constraints that might also delay plasmid production. For example, some DNA sequences are simply difficult to work with. These sequences might have too many Gs and Cs, be too repetitive, or they might have too much secondary or tertiary structure (knots and loops that DNA strands sometimes form). All these specificities can throw a wrench in each step of plasmid production, from design to sequencing. It is not uncommon for a tricky plasmid to consume over a month’s worth of time and effort; many perfectly good scientific ideas never come to fruition because plasmid production is difficult.    

While we often have a propensity to plan for perfect execution, a wise and experienced biologist will often cite the 3x Rule to their dewy-eyed students: everything takes three times as long and for three times the cost. 

For this reason, there are also many tricks that a scientist dedicated to speed will employ to accelerate this pipeline. For example, I remember heading into lab early in the morning to pick colonies so that the 6 milliliter liquid E.coli cultures might be just turbid enough (indicating cell growth and division) to harvest the DNA and hand it off to the sequencing facility that same evening. Other scientists might not PCR amplify DNA fragments, but take an aliquot of DNA straight from the precious green-capped tube and directly input it into the assembly reaction. Some extreme scientists will skip ordering DNA fragments all together — a seven day time sink — and assemble oligos, which are short pieces of single stranded DNA and typically ship next-day.

When I asked a dozen biologists how much time and money it would take to create a 12 kilobase plasmid from scratch, answers ranged from $1,000 in two weeks to $10,000 in a month, and everywhere in between. Each lab, and even each scientist, has a preferred method of stitching DNA together. Because the assembly step is so bespoke and nuanced, outsourcing the process to specialists seems like an intuitive solution, yet the average synthetic biologist defaults to each of these three steps occurring at three different physical locations.