A novel framework for engineering protein loops exploring length and compositional variation.

Insertions and deletions (indels) are known to affect function, biophysical properties and substrate specificity of enzymes, and they play a central role in evolution. Despite such clear significance, this class of mutation remains an underexploited tool in protein engineering with few available platforms capable of systematically generating and analysing libraries of varying sequence composition and length. We present a novel DNA assembly platform (InDel assembly), based on cycles of endonuclease restriction digestion and ligation of standardised dsDNA building blocks, that can generate libraries exploring both composition and sequence length variation. In addition, we developed a framework to analyse the output of selection from InDel-generated libraries, combining next generation sequencing and alignment-free strategies for sequence analysis. We demonstrate the approach by engineering the well-characterized TEM-1 ß-lactamase Ω-loop, involved in substrate specificity, identifying multiple novel extended spectrum ß-lactamases with loops of modified length and composition-areas of the sequence space not previously explored. Together, the InDel assembly and analysis platforms provide an efficient route to engineer protein loops or linkers where sequence length and composition are both essential functional parameters.

Selection platforms for directed evolution in synthetic biology.

Life on Earth is incredibly diverse. Yet, underneath that diversity, there are a number of constants and highly conserved processes: all life is based on DNA and RNA; the genetic code is universal; biology is limited to a small subset of potential chemistries. A vast amount of knowledge has been accrued through describing and characterizing enzymes, biological processes and organisms. Nevertheless, much remains to be understood about the natural world. One of the goals in Synthetic Biology is to recapitulate biological complexity from simple systems made from biological molecules-gaining a deeper understanding of life in the process. Directed evolution is a powerful tool in Synthetic Biology, able to bypass gaps in knowledge and capable of engineering even the most highly conserved biological processes. It encompasses a range of methodologies to create variation in a population and to select individual variants with the desired function-be it a ligand, enzyme, pathway or even whole organisms. Here, we present some of the basic frameworks that underpin all evolution platforms and review some of the recent contributions from directed evolution to synthetic biology, in particular methods that have been used to engineer the Central Dogma and the genetic code.

Raman spectroscopy and chemometrics for on-line control of glucose fermentation by Saccharomyces cerevisiae.

This work presents the use of Raman spectroscopy and chemometrics for on-line control of the fermentation process of glucose by Saccharomyces cerevisiae. In a first approach, an on-line determination of glucose, ethanol, glycerol, and cells was accomplished using multivariate calibration based on partial least squares (PLS). The PLS models presented values of root mean square error of prediction (RMSEP) of 0.53, 0.25, and 0.02% for glucose, ethanol and glycerol, respectively, and RMSEP of 1.02 g L(-1) for cells. In a second approach, multivariate control charts based on multiway principal component analysis (MPCA) were developed for detection of fermentation fault-batch. Two multivariate control charts were developed, based on the squared prediction error (Q) and Hotelling's T(2) . The use of the Q control chart in on-line monitoring was efficient for detection of the faults caused by temperature, type of substrate and contamination, but the T(2) control chart was not able to monitor these faults. On-line monitoring by Raman spectroscopy in conjunction with chemometric procedures allows control of the fermentative process with advantages in relation to reference methods, which require pretreatment, manipulation of samples and are time consuming. Also, the use of multivariate control charts made possible the detection of faults in a simple way, based only on the spectra of the system.