Rapid hierarchical assembly of medium-size DNA cassettes

Jonathan Leo Schmid-Burgk 2 3 Zhen Xie 1 2 3 Stefan Frank 0 2 Sebastian Virreira Winter 0 2 Sibylle Mitschka 0 2 Waldemar Kolanus 0 2 Andrew Murray 2 3 4 Yaakov Benenson 2 3 0 Program Unit Molecular Immune and Cell Biology, Laboratory of Molecular Immunology, LIMES (Life and Medical Sciences Institute), University of Bonn , Carl-Troll-Strae 31, 53115 Bonn, Germany 1 Department of Biological Engineering, Massachusetts Institute of Technology , 40 Ames St, Cambridge, MA 02142, USA 2 Present addresses: Jonathan Leo Schmid-Burgk, Unit for Clinical Biochemistry, Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital, University of Bonn , Sigmund-Freud-Strae 25, 53127 Bonn , Germany Zhen Xie, Center for Synthetic and Systems Biology , TNLIST, Tsinghua University , Beijing , 100084, China Stefan Frank, Department of Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Human Stem Cell Pluripotency Group , Von-Esmarch-Str. 54, 48149 Mu nster Sebastian Virreira Winter, Department of Cellular Microbiology, Max Planck Institute for Infection Biology , Charite platz 1, 10117 Berlin , Germany Yaakov Benenson, Department of Biosystems Science and Engineering , Eidgeno ssische Technische Hochschule (ETH) Zu rich , Mattenstrasse 26, 4058 Basel, Switzerland 3 FAS Center for Systems Biology, Harvard University , 52 Oxford St, Cambridge, MA 02138, USA 4 Department of Molecular and Cell Biology, Harvard University , 52 Oxford St, Cambridge, MA 02138, USA Synthetic biology applications call for efficient methods to generate large gene cassettes that encode complex gene circuits in order to avoid simultaneous delivery of multiple plasmids encoding individual genes. Multiple methods have been proposed to achieve this goal. Here, we describe a novel protocol that allows one-step cloning of up to four gene-size DNA fragments, followed by a second assembly of these concatenated sequences into large circular DNA. The protocols described here comprise a simple, cheap and fast solution for routine construction of cassettes with up to 10 gene-size components. - Construction of large gene cassettes that encode entire gene circuits is in acute demand in synthetic biology. Traditional cloning techniques make this process extremely laborious due to the step-wise nature of these protocols and the increasing dearth of unique restriction sites as the constructs become larger. As a result, the last two decades have seen the search for more efficient methods, with the first major advance being the invention of Ligase-independent cloning (LIC) (14). That method circumvented the use of restriction sites by generating long, unique single-stranded overhangs using the 30!50 exonuclease activity of T4 DNA polymerase in combination with flanking dsDNA termini lacking one of the four nucleotides (chew-back) (Figure 1). The original report demonstrated the ligation of an insert into a vector, i.e. the complexity of the process did not go beyond traditional restrictionligation cloning. To the best of our knowledge, the first attempt to combine three fragments with long ssDNA overhangs was described by Donahue et al. (5). However, generating the overhangs was enabled by including ribose residues in the PCR primers, which would not allow a hierarchical assembly of larger constructs without recurrent PCR steps since the ribose residues are lost after bacterial amplification. Performing PCRs after each assembly step is unfavourable when the assembly intermediates reach a certain size and it may introduce additional mutations. A different strategy was shown in a report by GeuFlores et al. (6), where overhangs were generated by uracil excision-based cloning. Four fragments were assembled in a single step; however, the requirement to include dU residues in the primers results in the same PCR dependency, which is even further restricted to a special DNA polymerase. A number of breakthroughs in high-throughput DNA assembly were reported in the context of whole-genome synthesis by Gibson et al. Two alternative methods were put forward. The first is an extension of LIC, with a major difference being the non-specific chew-back of overlapping DNA termini, and the reliance on DNA repair machinery of the bacterial host to deal with imperfect annealing of the resulting overhangs. Overlaps of at least 40 bp were shown to allow assembly of up to four fragments in a single cloning reaction (7,8). Another feature of the method is the hierarchical assembly, where the cloning vectors that contain the assembled fragments contain NotI restriction sites that are used to excise the combined sequences for the next cloning level. While the method is highly efficient in producing very long DNA from synthetic starting materials of a few kilobases, it is unclear whether the protocols could efficiently assemble shorter building blocks of fewer than 1000 base pairs due to the risk of complete DNA degradation by nonspecific chew-back. Besides, generating overhangs of 40 bp in a PCR reaction requires relatively expensive primers of at least 60 nt in length. Moreover, including additional functional sequences in the primers, a common practice in recombinant DNA work, can easily push the total primer length to 100 nt. The second method recently shown by the same group demonstrates concurrent assembly of up to 25 DNA fragments in yeast using recombination of overlapping DNA termini (9). While being a tour-de-force of highthroughput assembly, a few features of the process might pose problems in gene circuit assembly. First, the overlaps are at least 80 bp long and thus may not be readily introduced via PCR primers. Second, a sequence that appears more than once in different building blocks (such as a common promoter) could lead to undesirable recombination and a compromised final product. The method we describe here uses short overlaps of 20 bp and specific chew-back to accomplish hierarchical assembly of about 10 gene-size DNA fragments in a two-step process. MATERIALS AND METHODS Primers were phosphorylated in 20 ml reactions containing 5 mM primer, 1 PNK buffer (NEB), 1 mM ATP and 8 U PNK for 1 h at 37 C. The enzyme was heat-inactivated for 20 min at 65 C. PCR amplification was performed according to the manufacturers protocol with either Pfu Ultra II Fusion HS (Inverter circuit, reprogramming circuits 1 and 2); KOD extreme DNA polymerase (for the CAG promoter containing amplicons in reprogramming circuits) or with Phusion DNA polymerase (reprogramming circuit 3). The heat-inactivated phosphorylation reactions were used as the source of primers without further purification. Where DpnI digest was performed, 50 ml of PCR reaction were mixed with 5.5 ml of Fermentas FastDigest Green buffer and 2 ml DpnI (Fermentas FastDigest). The reactions were incubated at 37 C for 1 h. Pfu Ultra II Fusion HS (Stratagene) A 100 ml reaction mix contained: 1 Pfu Ultra II buffer, 250 mM dNTPs each, 5 ng plasmi (...truncated)