Modular assembly of genes and plasmids using PCR-based and ribozyme-based gene engineering methods

The aim of this research was to develop simple methods that can be used to generate chimeric genes and gene libraries. The methods are designed to take advantage of the modular architecture of certain genes. By recombining functional modules from different genes, a modular gene can be created that encodes a novel protein that performs a particular desired function. For example, this approach can be used to develop novel protein therapeutics, or chimeric biosynthetic enzymes that produce therapeutically relevant small molecules.; A new technique is described here that allows for the creation of recombinant DNA molecules by the seamless assembly of Polymerase Chain Reaction (PCR) products. Genes assembled using this method can be cloned directly in E. coli in a ligation-independent fashion. This technique was used to assemble a set of customized expression vectors from a set of interchangeable modular vector elements (i.e., selectable marker genes and replication origins). This modular vector system produced 18 unique plasmid vectors.; Experiments were also performed to develop RNA recombination methods that allow “module shuffling” to occur at the RNA level both in vivo and in vitro. A ribozyme-based gene assembly system was developed that used trans-splicing to assemble chimeric RNAs in either a directed or stochastic fashion. Such chimeric RNAS can be directly expressed in vivo or cloned following in vitro assembly.; Studies reported here demonstrate for the first time trans-splicing of the LtrB intron from the bacterium Lactococcus lacti. Trans-splicing of a cloned intron was observed both in vitro , and in vivo in E. coli. In addition, experiments were performed that used the LtrB intron along with the yeast aI5γ intron to perform the first non-naturally occurring three-molecule group II intron trans-splicing reaction.; The technologies described here provide useful alternatives to standard gene engineering techniques. The PCR cloning technique described here has been demonstrated to be an effective tool for site-directed mutagenesis. In addition, this ligation-independent cloning method is well suited for automated sample handling and should allow for rapid assembly of large sets of chimeric genes.