BioBrick-based 'Quick Gene Assembly' in vitro

Abstract

Because of the technological limitations of de novo DNA synthesis in (i) making constructs containing tandemly repeated DNA sequence units, (ii) making an unbiased DNA library containing DNA fragments with sequence multiplicity in a specific region of target genes, and (iii) replacing DNA fragments, development of efficient and reliable biochemical gene assembly methods is still anticipated. We succeeded in developing a biological standardized genetic parts that are flanked between a common upstream and downstream nucleotide sequences in an appropriate plasmid DNA vector (BioBrick)-based novel assembly method that can be used to assemble genes composed of 25 tandemly repeated BioBricks in the correct format in vitro. We named our new DNA part assembly system: 'Quick Gene Assembly (QGA)'. The time required for finishing a sequential fusion of five BioBricks is less than 24 h. We believe that the QGA method could be one of the best methods for 'gene construction based on engineering principles' at the present time, and is also a method suitable for automation in the near future.

Keywords: BioBrick; engineering principle; gene assembly; gene designing; magnetic beads.

Figure 1.

Schematic representation of the QGA protocol. (a) Preparation of a DNA fragment containing a BioBrick part. P: prefix DNA fragment, BB: BioBrick, S: suffix DNA fragment, 100 bp UP-F: 100 base pair upstream forward primer, 200 bp DN-R: 200 base pair downstream reverse primer, circled P: phosphate residue, X: XbaI restriction site. (b) Structural change of extending the DNA molecule in each step of QGA. MAGB: magnetic beads, E: EcoRI restriction site, S: SpeI restriction site, S/X: mixed restriction site (scar), TTT…TTT: oligo-dT conjugated to magnetic beads, AAA…AAA: oligo-dA in DNA adaptor.

Figure 2.

Size analysis of PCR products after an increase in the number of QGA cycles. (a) Predicted assembled structures of products after increase of cycles of QGA. Cycle numbers of QGA and predicted sizes of assembled DNA fragments after each cycle are indicated at the left of each construct. PtetR: promotor BioBrick of tetracycline resistance gene. (b) Distribution of amplified DNA fragments by PCR when assembled DNA fragments after each cycle were used as a template. QGA cycle numbers are indicated above each lane. Copy numbers of PtetR fragments are indicated at the right side of each band.

Figure 3.

Negative effects on the QGA process resulting from the change of each condition away from optimum conditions. Lane 1: assembled products under optimum conditions. Lane 2: replacement of the restriction enzyme from SpeI-HF (NEB) to SpeI (NEB). Lane 3: changing of ligation condition from ‘at 16 °C for 30 min’ to ‘at 25 °C for 10 min’. Lane 4: removal of 0.1% Triton X-100 from the rinsing solution. Lane 5: removal of 0.6 M trehalose from the digestion premix. Lane 6: combination of all changes tested from lanes 2–5. Copy numbers of PtetR fragment are indicated at the right side of each band.

Figure 4.

Negative effects of enzyme solution mixing from pipetting up and down compared to the agitation method. Distribution of amplified DNA fragments by PCR when assembled DNA fragments were used as a template after each cycle. Cycle numbers of QGA are indicated above each lane. Copy numbers of PtetR fragments are indicated at the right side of each band.

Figure 5.

Effects of the increase of BioBrick length in QGA. Distribution of amplified DNA fragments by PCR (length of each PCR product containing 5, 10, 15, 20, and 25 tandem repeats of PtetR is 514 bp, 814 bp, 1114 bp, 1414 bp, and 1714 bp, respectively) when assembled DNA fragments were used as a template after each cycle. Cycle numbers of QGA correspond to lane numbers. Copy numbers of PtetR fragments are indicated at the right side of each band.

Figure 6.

Assembly of functional genes from distinct BioBricks. (a) Schematic representation of the QGA process from a single DNA construct to the finished complete gene assembly is shown together with their lengths. P: prefix DNA fragment, PtetR: TetR promotor, RBS: ribosome binding site, GFP: DNA fragment encoding green fluorescent protein (BBa_I13500), RFP: DNA fragment encoding red fluorescent protein (BBa_K093005), dT: double terminator (BBa_B0015), S: suffix DNA fragment. (b) Sizes of PCR products amplified from assembled genes. White arrowheads indicate the expected DNA fragments. The numbers above each lane are corresponding to the numbers shown in panel A. (c) Images of colonies on agarose LB plates containing 100 μg/mL ampicillin. Expression of GFP and RFP was monitored under white light. Scale bars are 2 mm.