Golden Gate assembly¶

A Type IIS enzyme cuts outside its own site, so each fragment can be cut to a four-base overhang you choose, and every fragment joined in one tube in a fixed order. One command picks the enzyme, places the overhangs, designs the primers that carry the tails, builds the plasmid you should get, and writes the bench protocol.

When to use it¶

• Two to about a dozen fragments, joined in a defined order, in one reaction.
• You want to choose what the junction reads, down to the base, and leave no scar you did not pick.
• No part holds a site for one of the Type IIS enzymes, or you will let a silent codon change clear the sites that are there.

Use Gibson instead when the junctions may gain a few bases and you would rather not think about enzymes. Use restriction and ligation when the two plasmids already share a usable pair of sites.

What you need¶

One file for the vector, and one for each insert, named in the order they go round the product. SnapGene .dna, GenBank and FASTA all read. The run below uses two files that ship with the repo.

Run it¶

pixi run mbio cloning goldengate plan tests/data/pUC19.dna tests/data/GFP.dna --out plan/

It prints the design in one line, then the nine files it wrote:

pUC19-GFP: 3347 bp, BbsI, 2 fragments, overhangs ATGA, TGGC, fidelity 100% (measured), checks warn
plan/product.dna
plan/puc19-backbone-amplicon.dna
plan/gfp-amplicon.dna
plan/primers.tsv
plan/protocol.json
plan/puc19-backbone-amplicon-map.html
plan/gfp-amplicon-map.html
plan/product-map.html
plan/protocol.html

Read that line before anything else. It chose BbsI because BsaI reads a site in both pUC19 and GFP, and BsmBI reads two in pUC19; cutting with either would cut the parts as well as the ends, and BbsI cuts neither. Where no enzyme is free, the command stops and says so. It never changes your sequence unless you ask.

fidelity 100% (measured) is how well those two overhangs should pair, from ligation data measured on BbsI itself. checks warn means nothing failed, but something wants a look.

What it wrote¶

Each name links to what that run wrote, published here unedited. Nothing in it is typed by hand, so the same two input files always give the same nine. To change the design, change the command and run it again. To change what the page says, edit protocol.json and make the page from it again:

pixi run mbio protocol render plan/protocol.json

How to read the protocol¶

Open protocol.html. The top states the whole design in eight cards and four sentences. Here they say GFP goes in on the opposite strand from the lac promoter, that nothing is annotated ahead of GFP to start translation, so the clone is not expected to glow, and that the insertion breaks lacZα, so correct clones are white. All four are read off the finished plasmid, so they cannot disagree with the map.

Under them sit the badges. primers is amber here: of nine oligos, two carry a warning and none fails. The oligo sheet gives each oligo its own verdict in words, and a line under the sheet opens to show which check fired on which oligo.

Then eleven numbered steps, from the two PCRs to sequencing. Reaction tables rescale when you change the number of reactions. Gel steps draw the bands to expect: at the screen, a correct clone gives 160 and 838 bp and an empty vector gives 177 bp, so read the big band first.

Before you order¶

• Read the protocol once, top to bottom.
• Look at every badge that is not green, and decide about it.
• Open the line under the oligo sheet and decide about each warning.
• Check the product map holds what you meant to clone.
• Check the screening lanes can still be told apart.

Tips and troubleshooting¶

Every colony reads as empty vector. Either the plasmid template survived the DpnI digest, or the vector closed again. Check the 60 °C soak at the end of the program ran: it is a digest, not heat inactivation, and it cuts vector that never opened.

A warning sits on a primer with a tail. The junction fixes where that primer binds. The design tries every binding site the junction allows and keeps the one that warns least, so a warning left there is one nothing nearby could clear.

More fragments than about a dozen. A larger overhang set pairs less cleanly. The fidelity in the first line is the number to read before you order.

Where the numbers come from¶

Fidelity comes from ligation data measured for five enzymes. For any other enzyme one of those five stands in, and the protocol says which it used and that it is not specific. A ligase matrix you hold can be used instead, with --ligase-matrix; see ligation fidelity.

Volumes, units, times and temperatures are NEB's, cited step by step inside the protocol.

pixi run mbio cloning goldengate plan --help lists the rest: where the inserts go, which way round each one sits, holding a junction in frame, forcing an enzyme, and which polymerase and strain the protocol names.

References¶

• Engler, C., Kandzia, R. and Marillonnet, S. (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS ONE 3(11): e3647.
• Pryor, J.M., Potapov, V., Kucera, R.B., Bilotti, K., Cantor, E.J. and Lohman, G.J.S. (2020) Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design. PLoS ONE 15(9): e0238592.
• NEB, NEBridge Golden Gate Assembly Kit (BsaI-HFv2) instruction manual, NEB #E1601S/L, version 5.0_6/26.
• NEB, Protocol for NEBridge Ligase Master Mix (NEB #M1100).