Modular cloning: a transcription unit from level-0 parts

• You will end with: a 1,702 bp circular level-1 construct from three parts, then four constructs (1,702, 1,762, 1,722 and 1,782 bp) from one plan.
• Time: about 25 minutes.
• Files: MC-L1-Destination.gb, MC-L0-Promoter-A.gb, MC-L0-Promoter-B.gb, MC-L0-CDS-A.gb, MC-L0-CDS-B.gb, MC-L0-Terminator.gb, MC-wrong-position.gb. Every file is a synthetic example (invented sequence).
• Before this: Golden Gate: one gene into one vector, and Before you start if the Bench or the sample files are new to you.

A promoter, a coding part and a terminator, each made for its own position in the MoClo standard, go into a level-1 destination vector in one reaction. The reaction is the Golden Gate one; this page only adds the standard on top of it. Words you meet are explained at the end under How it works.

The parts, as the files have them (lengths are of the whole file, flanks included):

The destination is circular and the parts are linear. BsaI cuts the destination twice and every part twice; no other Type IIS enzyme in the bundled set cuts any file.

Exercise 1 of 3: one transcription unit

1. Choose File ▸ Open and select MC-L1-Destination.gb, MC-L0-Promoter-A.gb, MC-L0-CDS-A.gb and MC-L0-Terminator.gb (or drag them onto the window).
2. Close every other tab.
3. Click the Bench tab.
4. At the top of the middle column, click Modular.

   You see the heading Modular cloning, with a standard, a plan beside it.

5. Set Standard to MoClo plant common syntax, and leave and at no other.

   You see the paper it comes from named (Weber et al. 2011; Patron et al. 2015), and Enzyme reading BsaI.

   Why

   The parts were cut for BsaI; the enzyme must be the one that makes their overhangs, and the standard starts it on BsaI.

6. Look at the parts list under the enzyme pickers.

   You see the four documents, each with its tick box ticked.

   If instead you see more than four documents, close the extra tabs (or untick them): the panel puts every open document in the plan, and an extra part changes the combinations.

7. Under Positions, read the menu beside each document.

   Document	Menu reads
   pMC-L1-Destination	Detected: Destination
   MC-L0-PromA	Detected: Promoter + 5′UTR
   MC-L0-CdsA	Detected: CDS
   MC-L0-Term	Detected: 3′UTR + terminator

   Why

   The panel digests each document and reads the overhangs of the piece BsaI releases. You never tag a part by hand unless it cannot find one.

8. Below that, read the plan. Each slot's right overhang meets the next slot's left one, and the last meets the destination's first; a slot that met nothing would be reported here, before the reaction.

   You see four slots, destination first, then in position order: Destination CGCT-GGAG · pMC-L1-Destination, Promoter + 5′UTR GGAG-AATG · MC-L0-PromA, CDS AATG-GCTT · MC-L0-CdsA, and 3′UTR + terminator GCTT-CGCT · MC-L0-Term.

9. Read the summary line above the products.

   1 combination: 1 assemble.

   The one product is named:

   pMC-L1-Destination + MC-L0-PromA + MC-L0-CdsA + MC-L0-Term

   and its detail line reads:

   1,702 bp circle

   You see no overhang warning.

   Why

   The sizes add up less than you might expect: the backbone BsaI leaves is 1,254 bp, the parts 74, 304 and 70 bp, and 1,254 + 74 + 304 + 70 = 1,702. Each junction's four overhang bases are in the product once, not twice, one from each neighbour. On the map, one base, the A of AATG, lies between the end of synthetic promoter A and the start of synthCDS A, which begins on the ATG. And GCTT follows the stop codon directly and starts synthetic terminator: the four bases are the junction, not extra sequence.

10. Click Assemble beside the product. It opens the product as a new tab, with the parts it was made from in its history.

    You see a new tab, pMC-L1-Destination+MC-L0-PromA+MC-L0-CdsA+MC-L0-Term assembly.

    Checkpoint:

    • The product is 1,702 bp, circular, with AmpR, ori, synthetic promoter A, synthCDS A and synthetic terminator, and no lacZ-dropout.

    If not, or if the tab name lists more parts than these three, the tube held extra tabs: close them all but the four documents and assemble again.

11. Open the product's Features tab.

    You see the three parts in order on the strand: promoter, then synthCDS A, then terminator.

12. Open the product's Enzymes tab.

    You see BsaI ticked (the Bench's enzyme, as the product opens) with 0 sites in the product, so nothing is marked: the vector had 2, and the cut threw them away with the dropout.

13. Optional: translate synthCDS A from its ATG (the sidebar's Translate tab). It is one open reading frame of 100 residues and a stop (303 bp), starting on the ATG of the AATG junction.

Exercise 2 of 3: one part at the wrong position

MC-wrong-position.gb (document MC-L0-YTK-Type3, 106 bp) is a part cut for position Type 3 of the Yeast Toolkit: overhangs TATG...ATCC. It is valid in YTK, and has no place in MoClo plant syntax.

1. Choose File ▸ Open and select MC-wrong-position.gb, so it is open with the four documents of Exercise 1.
2. In the panel, look at its menu under Positions.

   Detected: none found

   and a note:

   Not placed: MC-L0-YTK-Type3. Their ends match no position of MoClo plant common syntax with BsaI; set one by hand, or leave them out.

   You see the summary line still reading 1 combination: 1 assemble., because an unplaced part is not in the plan and cannot break it.

   Why

   The panel only places a part whose two overhangs are one position's. Anything else, a part from another kit or one with a typo in an overhang, stays out and is named.

3. Open that menu and choose CDS AATG-GCTT to force it.

   You see the slot CDS listing both coding parts, and the summary line reading 2 combinations: 1 assemble, 1 cannot form.

4. Read the cannot-form row (cannot-form rows come first).

   pMC-L1-Destination + MC-L0-PromA + MC-L0-YTK-Type3 + MC-L0-Term

   Its detail line, where the size would be, reads:

   No part starts with the overhang BsaI 5′ AATG left by MC-L0-PromA. 2 of 4 parts were never reached.

   You see no Assemble button on that row.

   Why

   The reaction is the real one. The promoter hands on AATG and this part starts with TATG, so nothing can join it. This is what a position guarantees: if the overhangs fit, it assembles.

5. Open the menu again and choose Detected: none found (or close the tab) before the next exercise.

Exercise 3 of 3: two promoters by two coding parts

A combinatorial plan: put two alternatives in a slot and the panel runs every combination.

1. Close MC-wrong-position.
2. Choose File ▸ Open and add MC-L0-Promoter-B.gb (MC-L0-PromB, 116 bp, synthetic promoter B) and MC-L0-CDS-B.gb (MC-L0-CdsB, 386 bp, synthCDS B), with the first exercise's four documents still open.

   If instead you see more than six documents in the parts list, close the extra tabs before you assemble: every open document goes into the plan, and extra parts make extra combinations.

3. Read the menus under Positions.

   You see Detected: Promoter + 5′UTR for both promoters and Detected: CDS for both coding parts.

   Why

   They are cut for the same positions as the first two, so they are alternatives to them.

4. Read the plan's slots.

   You see GGAG-AATG · MC-L0-PromA, MC-L0-PromB and AATG-GCTT · MC-L0-CdsA, MC-L0-CdsB, with the destination and terminator slots as before.

5. Read the summary line and the four rows, in this order.

   4 combinations: 4 assemble.

   Product	Detail
   pMC-L1-Destination + MC-L0-PromA + MC-L0-CdsA + MC-L0-Term	1,702 bp circle
   pMC-L1-Destination + MC-L0-PromA + MC-L0-CdsB + MC-L0-Term	1,762 bp circle
   pMC-L1-Destination + MC-L0-PromB + MC-L0-CdsA + MC-L0-Term	1,722 bp circle
   pMC-L1-Destination + MC-L0-PromB + MC-L0-CdsB + MC-L0-Term	1,782 bp circle

   Why

   2 x 2 x 1 x 1. The lengths differ by the parts: promoter B is 20 bp longer than A, CDS B is 60 bp longer than CDS A.

6. Click Assemble all 4. It opens every product that forms, one tab each (up to 24 at a time; a plan runs at most 500 combinations, so untick parts of a bigger one).

   You see four new tabs named like the first, for example pMC-L1-Destination+MC-L0-PromB+MC-L0-CdsA+MC-L0-Term assembly.

   Checkpoint:

   • Four new tabs, each circular, at 1,702, 1,762, 1,722 and 1,782 bp, in that order.

   If not, or if you see more than four new tabs, the tube held extra tabs: close the extras and assemble again.

7. Open a product's Features and Enzymes tabs.

   You see AmpR, ori, one promoter (A or B), one coding part (A or B) and synthetic terminator, never both promoters or both coding parts, and BsaI ticked with 0 sites.

Done when:

• The first product is 1,702 bp, circular, with synthetic promoter A, synthCDS A and synthetic terminator.
• The four products are 1,702, 1,762, 1,722 and 1,782 bp, and the panel showed no overhang warning (GGAG, AATG, GCTT and CGCT are four distinct overhangs, none one base from another).

Next: back to the tutorials index. Numbers off? See If a number does not match.

How it works

The words used above:

• Standard: an agreed list of positions (Promoter, CDS, Terminator...), each with the two overhangs a part for it is cut with. This page uses the MoClo plant common syntax, one of the two standards bundled with PlasmidPop (the other is the Yeast Toolkit, YTK). The panel carries only the overhang sequences, which are published facts; it holds no parts.
• Level 0 part: one genetic element (a promoter, a coding sequence, a terminator) with the overhangs of its position, released from its plasmid by BsaI. Here each part is a small linear file: the part, a BsaI site at each end pointing inwards, and a few bases of flank.
• Level 1: the reaction that joins level-0 parts, in position order, into one transcription unit in a destination vector. The destination's two BsaI sites sit inside a lacZ-dropout and point outwards, so cutting throws the dropout away and leaves a backbone whose two ends take the first and the last position.
• Overhang: the 4 single-stranded bases a cut leaves. Two ends join only when their overhangs match.
• Position: where a part goes in the unit. The panel reads it from the part's own overhangs.
• Plan: one slot per position that has a part, plus the destination. Two parts in one slot are alternatives, and the plan makes every way of choosing one per slot.

The standard's three positions

MoClo plant common syntax has more positions than this page uses (a split promoter and 5′UTR, N- and C-terminal parts of a CDS). The three used here, with the overhangs BsaI leaves on a part for each:

The right overhang of one position is the left overhang of the next, so the parts can only chain in one order: GGAG, promoter, AATG, CDS, GCTT, terminator, CGCT. The destination's backbone ends are CGCT and GGAG, which closes the ring. That is the whole idea of the standard: because every promoter is cut with GGAG...AATG, any promoter fits any CDS, and parts made by different people interchange. A part cut with the wrong overhangs does not belong at a position, and the panel will not place it there (Exercise 2). The ATG that ends AATG is the start codon of the CDS.

Two levels, one tutorial: here the level-0 parts are joined into a level-1 unit. A real kit then joins several level-1 units into a level-2 construct with a second enzyme (BpiI/BbsI, so that the BsaI sites left in the level-1 vectors are not cut). The panel plans one level at a time; open the level-1 product, choose the other enzyme on the Bench, and the same steps do the next level. That is not done here.