GoldBricks: an improved cloning strategy that combines features of Golden Gate and BioBricks for better efficiency and usability

Abstract

With increasing complexity of expression studies and the repertoire of characterized sequences, combinatorial cloning has become a common necessity. Techniques like BioBricks and Golden Gate aim to standardize and speed up the process of cloning large constructs while enabling sharing of resources. The BioBricks format provides a simplified and flexible approach to endless assembly with a compact library and useful intermediates but is a slow process, joining only two parts in a cycle. Golden Gate improves upon the speed with use of Type IIS enzymes and joins several parts in a cycle but requires a larger library of parts and logistical inefficiencies scale up significantly in the multigene format. We present here a method that provides improvement over these techniques by combining their features. By using Type IIS enzymes in a format like BioBricks, we have enabled a faster and efficient assembly with reduced scarring, which performs at a similarly fast pace as Golden Gate, but significantly reduces library size and user input. Additionally, this method enables faster assembly of operon-style constructs, a feature requiring extensive workaround in Golden Gate. Our format allows such inclusions resulting in faster and more efficient assembly.

Keywords: BioBricks; Golden Gate; Type IIS enzymes; bacteria; chloroplast; operon cloning.

Figure 1.

Advantage of Type IIS ligation: sticky ends of Type IIS enzymes can be designed to be non-palindromic, which prevents the issue of self-ligation responsible for several unintended byproducts.

Figure 2.

A basic scheme for defining a GoldBrick part and a three-part assembly: (A) Here each part has a four-enzyme flank and part is typified by the overhang sequence of the Type IIS site. Each part has specific compatibility for the suffix and the prefix part. For an example of the three-part assembly, the parts can be excised using sites marked in the box and subsequently ligated to give a strictly ordered product. If the destination vector has different antibiotic marker, parts do not need to be purified for assembly. (B) Sequence format to show directionality of Type IIS enzymes.

Figure 3.

Demonstration of the three-part assembly: (A) A three-part assembly was performed using parts U1, CDS1 and U2 that were 62 bp, 309 bp and 112 bp long, respectively. After transformation, eight colonies were selected randomly and DNA was prepared. (B) DNA was digested using PagI, ScaI and MauBI, which would give bands of the mentioned sizes from successful assemblies. The agarose gel of the digested DNA of empty pStart-IV (lane C) and eight randomly selected colonies (lanes 1–8) demonstrates that all clones harbored the correct assembly. Specific fragment sizes are marked in blue.

Figure 4.

Demonstration of the five-part assembly: (A) Schematic for two assemblies 5P-A and 5P-B is shown. Each part was cut in its holding vector using the enzyme shown in the figure (N = NotI, Ec = Eco31I, Es = Esp3I and M = MauBI). Part ends that were dephosphorylated are marked by *. These five parts were then ligated into pStart-IV and selected on kanamycin. (B) Six colonies that were PCR positive for inserts were randomly picked and DNA prepared. These were validated for correct assembly using restriction digestion according to the schematic. All colonies showed the correct insertion. Specific fragment sizes are marked in blue.

Figure 5.

Assembly extension: Level 1 assemblies 5P-A, 5P-B and a tobramycin resistance cassette were combined using the restriction enzyme scheme shown in (A). Sites marked with ‘*’ were dephosphorylated. Six random clones of Super Assembly A (SA-A) were checked using restriction digestion according to schematic (B) and all were found to have correct assemblies. Specific fragment sizes are marked in blue.