Proof-of-concept assembly of 16TU construct.

<p>(A) A schematic showing the four intermediate Level 2 constructs for the assembly of the 16-TU construct. The carotenoid biosynthesis genes <i>crtE</i>, <i>crtB</i>, <i>crtI</i>, and <i>crtY</i> assembled in the Vector A, the yellow chromoprotein genes <i>scOrange</i>, <i>amilGFP</i>, <i>amajLime</i>, and <i>fwYellow</i> in the Vector B, the pink chromoprotein genes <i>tsPurple</i>, <i>eforRed</i>, <i>spisPink</i>, and <i>mRFP1</i> in the Vector Γ, and the violacein biosynthesis genes <i>vioA</i>, <i>vioB</i>, <i>vioD</i> and <i>vioE</i> in the Vector Δ. (B) A schematic of the 16TU construct derived from the assembly of the four Level 2 cassettes, each containing 4-TUs, in the Level 1 Acceptor Vector A. (C) Cells transformed with the successfully assembled 16TU construct grew into black colonies due to predominant colouring by protoviolaceinic acid. (D) Gel electrophoresis of six plasmids (isolated from the black colonies) digested with <i>PstI</i> and <i>EcoRI</i> resulting in bands of expected sizes—18.2kb for the insert and 2.2kb for the vector. (E) The same plasmids were digested with <i>PstI</i> and <i>AleI</i> resulting in the bands of expected sizes—7.1kb, 5.1 and 4.9kb (appear merged on the gel), and 3.2kb.</p

Reconstruction of the carotenoid biosynthesis transcriptional units.

<p>(A) A schematic of carotenoid biosynthetic pathway showing the enzymes mediating the production of zeaxanthin as the final product. (B) The multi-TU constructs made by Mobius Assembly to produce lycopene (<i>crtEBI</i>) and β-carotene (<i>crtEBIY</i>) (in Level 2 Acceptor Vectors) and for zeaxanthin (<i>crtEBIYZ</i>) by assembling <i>crtEBIY</i> and <i>crtZ</i> back in a Level 1 A Vector. Colonies producing lycopene are pink (C), β-carotene orange (D), and zeaxanthin yellow (E). Cells carrying intact Level 2 Vectors produced bright yellow colonies, and Level 1 Vectors pink colonies. Gel electrophoresis of the PCR from five colonies (pink, orange and yellow from each cloning, respectively) verified the correct size of the constructs; 4.3kb for lycopene (F), 5.7kb for β-carotene (G), and 6.7kb zeaxanthin (H). UV-Visible spectrophotometry showed expected peaks for lycopene (446nm, 472nm, and 503nm, I), β-carotene (450nm and 478nm, J) and zeaxanthin (450nm and 478nm, K).</p

Mobius Assembly framework.

<p>(A) Mobius Assembly uses a two-level (Level 1 and 2) approach for transcriptional unit (TU) and multi-TU augmentation. Each level is comprised of four Acceptor Vectors. The four Level 1 Acceptor Vectors (A, B, Γ, and Δ) carry <i>spisPink gene</i> as the visible cloning screening marker and confer Kanamycin resistance. The four Level 2 Acceptor Vectors (A, B, Γ, and Δ) carry <i>sfGFP gene</i> as the visible cloning screening marker and confer Chloramphenicol resistance. The standard parts stored in mUAVs are released and fused in a Level 1 reaction to form a TU. Up to four Level 1 TUs can be fused in a Level 2 reaction to form a multi-TU cassette. Switching back and forth between Level 1 and 2 leads to further expansion of multi-TUs according to the geometric sequence: 1, 4, 16, 64,…. Red arrows denote <i>Aar</i>I restriction sites and Purple arrows <i>Bsa</i>I restriction sites. (B, C, and D) <i>E</i>. <i>coli</i> colonies carrying mUAV (B), Level Acceptor 1 Vector A (C), and Level 2 Acceptor Vector A (D), which respectively exhibit purple, magenta and yellow colour after overnight incubation. Successful assembly produces white colonies.</p

Mobius Assembly standard part generation.

<p>(A) Mobius Universal Acceptor Vector (mUAV) is the vector which converts and hosts DNA fragments as standard parts. mUAV is flanked by the Type IIS restriction enzymes <i>Bsa</i>I and <i>Aar</i>I and carries <i>amilCP</i> gene as visible cloning screening marker. The inserts are amplified with primers containing <i>Aar</i>I recognition sites, the fusion sites with the mUAV, and the standard overhangs, and they replace <i>amilCP</i> cassette in a Golden Gate reaction. The standard parts are released by <i>Bsa</i>I digestion. E: <i>Eco</i>RI; P: <i>Pst</i>I. (B) Mobius Assembly embraces the 4bp standard part overhangs defined by MoClo, Golden Braid, and Phytobricks, to facilitate part sharing. The middle row illustrates the standard overhangs for major functional parts (promoter, coding sequence, and terminator); the top row shows the recommended overhangs for eukaryotic sub-functional parts, while the bottom row indicates ones for the prokaryotic counterparts.</p

Mobius Assembly vector toolkit.

<p>(A) The overhangs of the four Level 1 Acceptor Vectors. <i>BsaI</i> digestion releases the <i>spisPink</i> gene upon digestion to expose GGAG and CGCT, between which a TU will be incorporated. Each type of vector has unique overhangs at the 3’ end which guides the assembly of up to four TUs in a Level 2 Acceptor Vector. (B) Seven Auxiliary Plasmids provide End-to-End linkers and Middle-to-End linkers to assist Level 2 cloning. (C) The overhangs of the four Level 2 Acceptor Vectors. Digestion with <i>Aar</i>I releases the <i>sfGFP</i> gene and exposes GGAG and ACCC, between which up to four TUs will be fused into with the assistance of an Auxiliary Plasmid. 4A, 4B, 4Γ and 4Δ End-to-End linkers provide 5’ and 3’ overhangs and the missing Level 2 overhang when four Level 1 TUs are fused. Middle-to-End linkers 1, 2, and 3 are used when one, two or three Level 1 cassettes are fused in Level 2. They provide 5’ and 3’ overhangs and the CGCT overhang necessary for the cloning back to Level 1. (D) An example of how the Auxiliary Plasmids are used. A 7-TU construct is generated by combining the four TUs in the Level 2 Acceptor Vector A and the remaining three TUs in Vector B in a Level 1 reaction. Auxiliary Plasmid 4A is used for the four TUs in Acceptor Vector A, and the Auxiliary Plasmid 3 for the three TUs in Vector B. Red arrows demarcate <i>Aar</i>I restriction sites and purple arrows <i>BsaI</i> restriction sites.</p

Spatiotemporal plot and growth profiles for the BPM rings by pattern dependent expansion.

<p>A: Schematic of the modeling. B: Spatiotemporal plot for peak doubling by insertion. The value of reactant is represented by the gray scale. Each panel shows the first (C), second (D), third (E), and fourth (F) insertion. Each point indicate the middle point of segmented cell, then the color of points indicate the value of reactant <i>u</i>. Solid arrowheads indicate the points of peak insertion, and empty arrowheads are points of side branch generation.</p

Morphogenesis of <i>Rorippa aquatica</i> leaves.

<p>A, B: Mature leaf morphology of the simple leaf that was developed at 30°C (A) and the highly branched compound leaf that was developed at 20°C (B). Scale bar: 1 cm. C: Dissected shoot apex of a plant grown at 20°C, showing the nested group of leaf primordia with indented blade. D–F: Dissected primordial of a plant grown at 20°C for about 2 months. Each primodium has the 32th (D), 35th (E), and 39th (F) leaf primordium from the oldest (i.e. outermost) leaf of a plant. The larger leaf position numbers indicate younger leaves. Scale bar: 1 mm (C) and 200 µm (D–F). G: Comparison of the total number of leaflet primordial between experimentally observed and the theoretically estimated value.</p

Spatiotemporal plot and growth profiles for the BPM rings by Expansion inhibition.

<p>A: Spatiotemporal plot for peak doubling by splitting. The value of reactant is represented by the gray scale. Each panel shows the first (B), second (C), third (D), and fourth (E) splitting. Each point indicate the middle point of segmented cell, then the color of points indicate the value of reactant <i>u</i>. Solid arrowheads indicate the points of peak splitting, and empty arrowheads are points of side branch generation.</p

The numbers of leaflet primordia of each primary leaflet.

<p>A: Schematic of the branched structure of one half of a <i>R</i>. <i>aquatica</i> compound leaf. Circled numbers indicate the positions of primary leaflet (the horizontal axis in B), and theoretically derived recurrence formulas of each primary leaflet are shown by . The red numbers represent the numbers of leaflets formed on the 4^th primary leaflet (the vertical axis in B). B: A comparison between the experimentally observed data in actual plants and theoretically estimated numbers derived from mathematical formulae of leaflet on each primary leaflet. The magenta dots show the data from mature leaves. The number of leaflets at each stage was plotted as aligned at the center. The theoretical estimations are represented on a yellow planar graph, and the actual data in developing leaves as blue dots with columns.</p

Simulations of leaf primordia and branches.

<p>Each panel shows the simulated whole leaves (A–C) and primary leaflets (D–H). The simulated branches were crossover. Each branch was independently formed nested regular branches. The inserted number shows the time of iterative calculations (), and the arrowheads indicate each leaflet; filled, flamed, and dotted arrow heads represent the first, second, and third primary leaflet respectively.</p