Golden Gate in PlasmidPop: one gene into an expression vector

• You will end with: a 3,239 bp circular plasmid with synthGene between the promoter and the terminator (path A), or a 2,870 bp one with the insulin gene (path B).
• Time: about 20 minutes.
• Files: pGG-Expression.gb, GG-gene.gb (path A only).
• Before this: Before you start if the Bench or the sample files are new to you.

Before you click anything, close every tab you do not need: the Golden Gate simulation takes the other open tabs into the tube. Then choose one path. They are independent.

Path A: a synthetic ORF

GG-gene.gb is a plain 702 bp open reading frame, synthGene, ATG to stop, with no BsaI site and no cloning ends.

1. Choose File ▸ Open and select pGG-Expression.gb and GG-gene.gb (or drag both onto the window).

   If instead you see extra tabs, close them: the simulation would take them into the tube.

2. Click the pGG-Expression tab so it is in front.
3. Open the Primers tab in the sidebar and click Design primers….
4. Under Mode, click Clone an insert into this plasmid.
5. In step 3 of the panel, next to Ends, click Golden Gate part.

   You see pGG-Expression is the destination · 2,857 bp, and Enzyme stays BsaI.

6. In step 2 of the panel, What to insert, open Feature in another tab… and choose synthGene (CDS).

   You see synthGene in GG-gene, 702 bp.

7. Back in step 3, open Overhangs and choose From pGG-Expression: AATG…GCTT.

   You see both overhang fields filled in.

   Why

   The vector's own cut ends say which overhangs the gene needs.

8. Leave Include the start codon ticked and tick Include the stop codon.
   Why

   Nothing is fused after this gene (the terminator follows), so the stop codon stays. It is off by default because tags usually follow in frame.

9. Click Design primers.

   You see the part is 707 bp and the PCR product is 733 bp.

10. Click Simulate.

    You see PCR 733 bp · part 707 bp · product 3,239 bp and No warnings.

11. Look at the tab that opened, pGG-Expression-synthGene.

    You see a 3,239 bp circular product with synthGene and no lacZ-dropout.

    If not, close any extra tab (the simulation takes other open tabs that BsaI cuts into the tube) and click Simulate again.

12. Open its Enzymes tab and look at BsaI.

    You see BsaI ticked with 0 sites (the vector had 2): the cut threw them away.

The product is a scratch document; use File ▸ Save if you want to keep it.

Done when:

• The product is 3,239 bp and circular.
• synthGene sits between the promoter and the terminator, and there is no lacZ-dropout.

Path B: a gene from NCBI (human insulin, NM_000207)

The record is third-party data, so you fetch it; nothing in this folder contains it. It needs a network connection, and only the accession you type leaves the browser.

1. Choose File ▸ Open and select pGG-Expression.gb only.

   You see one tab, pGG-Expression.

   If instead you see other tabs, close them: the vector is the destination and stays in front, and the simulation takes other tabs into the tube.

2. Open the Primers tab in the sidebar and click Design primers….
3. Click Mode: Clone an insert into this plasmid.
4. In step 3 of the panel, next to Ends, click Golden Gate part.
5. In step 2 of the panel, What to insert, click Gene from NCBI….
6. Type NM_000207 and click Add. (This is the same search as File ▸ Open from NCBI…, with Add where that one says Open.)

   You see insulin preproprotein in the new tab, 333 bp.

7. In step 3, open Overhangs and choose From pGG-Expression: AATG…GCTT.
8. Leave Include the start codon ticked and tick Include the stop codon.

   You see the part is 338 bp and the PCR product is 364 bp.

9. Click Design primers.

   You see one amber caution: GC content above 65%.

   Why

   The amber GC caution is expected here (the gene's ends are GC-rich), and Simulate still works.

10. Click Simulate.

    You see a new tab, pGG-Expression-insulin preproprotein, a 2,870 bp circular product.

    If not, close any extra tab (the simulation takes other open tabs that BsaI cuts into the tube) and click Simulate again.

11. Open the Enzymes tab of the new tab.

    You see BsaI ticked, with 0 sites in the product.

Done when:

• The product is 2,870 bp and circular.
• The features are KanR, ori, synthetic promoter, INS with the insulin chain features and synthetic terminator, and there is no lacZ-dropout.

The oligos depend on the padding setting and on the annealing lengths the designer picks, so they are not written out. What does not depend on them is the part and the product length.

Next

Four parts and the warnings puts four parts in one tube in a scrambled tab order (3,118 bp, with a check digest, and an NCBI version), and Exercise 2, seeing the warnings shows three broken designs: a BsaI site inside a part, two parts that offer the same overhang, and two overhangs one base apart.

How it works

• Type IIS enzyme (BsaI here): cuts a short distance beside its recognition site, so the site is thrown away with the cut-off end.
• Overhang: the 4 single-stranded bases a cut leaves. Two ends join only when their overhangs match, so overhangs decide what goes where.
• Dropout: the stretch of the vector that the cut removes, with its two BsaI sites. The gene takes its place.
• Part: a piece with an overhang at each end, ready to go into a slot. Here the gene is the part, and its primers add the sites and overhangs.

pGG-Expression.gb is a circular 2,857 bp vector that already has a promoter and a terminator. Between them sit two BsaI sites around a lacZ-dropout. Cut with BsaI, the vector's backbone ends in AATG (after the promoter) and GCTT (before the terminator), which is the pair a MoClo plant CDS part uses. Your gene needs AATG before it and GCTT after it. PlasmidPop designs the two PCR primers that put them there, then simulates the reaction. You never type a sequence.

Every file is a synthetic example (invented sequence), and every number above is checked by src/core/cloning/goldenGateTutorial.test.ts, which runs these same files through the functions the app uses.

Background: Golden Gate on the Bench, and Golden Gate ends in the primer workspace.

Back to the tutorials index.