Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design.

DNA assembly is an integral part of modern synthetic biology, as intricate genetic engineering projects require robust molecular cloning workflows. Golden Gate assembly is a frequently employed DNA assembly methodology that utilizes a Type IIS restriction enzyme and a DNA ligase to generate recombinant DNA constructs from smaller DNA fragments. However, the utility of this methodology has been limited by a lack of resources to guide experimental design.

Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly.

Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzyme-dependent DNA assembly methods enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined by the ligation of Watson-Crick base-paired overhangs. However, ligation of mismatched overhangs leads to erroneous assembly, and low-efficiency Watson Crick pairings can lead to truncated assemblies.

DNA-dependent DNA polymerases.

This unit presents characteristics and reaction conditions of the DNA-dependent DNA polymerases, including E. coli DNA polymerase I and its Klenow fragment, T4 DNA polymerase, native and modified T7 DNA polymerase, phi29 DNA polymerase, Bst DNA polymerase, and Taq DNA polymerase. The unit also provides overviews of other classes of thermophilic DNA polymerases used in PCR applications (described fully in UNIT 15.

A view of consecutive binding events from structures of tetrameric endonuclease SfiI bound to DNA.

Many reactions in cells proceed via the sequestration of two DNA molecules in a synaptic complex. SfiI is a member of a growing family of restriction enzymes that can bind and cleave two DNA sites simultaneously. We present here the structures of tetrameric SfiI in complex with cognate DNA.

An asymmetric complex of restriction endonuclease MspI on its palindromic DNA recognition site.

Most well-known restriction endonucleases recognize palindromic DNA sequences and are classified as Type IIP. Due to the recognition and cleavage symmetry, Type IIP enzymes are usually found to act as homodimers in forming 2-fold symmetric enzyme-DNA complexes. Here we report an asymmetric complex of the Type IIP restriction enzyme MspI in complex with its cognate recognition sequence.

Changing the enzymatic activity of T7 endonuclease by mutations at the beta-bridge site: alteration of substrate specificity profile and metal ion requirements by mutation distant from the catalytic domain.

Phage-encoded resolvase T7 endonuclease I is a structure-specific endonuclease. The enzyme acts on a broad spectrum of substrates with a variety of DNA structures. The enzyme is a dimer with two separated catalytic domains connected by an elongated beta-sheet bridge.