The in vitro reconstitution of nucleosome and its binding patterns with HMG1/2 and HMG14/17 proteins

Using atomic force microscopy (AFM), the dynamic process of the in vitro nucleosome reconstitution followed by slow dilution from high salt to low salt was visualized. Data showed that the histone octamers were dissociated from DNA at 1M NaCl. When the salt concentration was slowly reduced to 650 mMand 300 mM, the core histones bound to the naked DNA gradually. Once the salt concentration was reduced to 50 mM the classic 'beads-on-a-string' structure was clearly visualized. Furthermore, using the technique of the in vitro reconstitution ofnucleosome,the mono- and di- nucleosomes were assembled in vitro with both HS2core (-10681 to -10970 bp) and NCR2 (-372to -194 bp) DNA sequences in the 5'flanking sequence of human b-globin gene. Data revealed that HMG 1/2 and HMG 14/17 proteins binding to both DNA sequences are changeable following the assembly and disassembly of nucleosomes. We suggest that the changeable binding patterns of HMG 14/17 and HMG1/2 proteins with these regulatory elements may be critical in the process of nucleosome assembly, recruitment of chromatin-modifying activities, and the regulation of human b-globin gene expression.

In vitro reconstitution guide for targeted synthetic metabolism of chemicals, nutraceuticals and drug precursors

With the developments in metabolic engineering and the emergence of synthetic biology,many breakthroughs in medicinal,biological and chemical products as well as biofuels have been achieved in recent decades.As an important barrier to traditional metabolic engineering,however,the identification of ratelimiting step(s)for the improvement of specific cellular functions is often difficult.Meanwhile,in the case of synthetic biology,more and more BioBricks could be constructed for targeted purposes,but the optimized assembly or engineering of these components for high-efficiency cell factories is still a challenge.Owing to the lack of steady-state kinetic data for overall flux,balancing many multistep biosynthetic pathways is time-consuming and needs vast resources of labor and materials.A strategy called targeted engineering is proposed in an effort to solve this problem.Briefly,a targeted biosynthetic pathway is to be reconstituted in vitro and then the contribution of cofactors,substrates and each enzyme will be analyzed systematically.Next is in vivo engineering or de novo pathway assembly with the guidance of information gained from in vitro assays.To demonstrate its practical application,biosynthesis pathways for the production of important products,e.g.chemicals,nutraceuticals and drug precursors,have been engineered in Escherichia coli and Saccharomyces cerevisiae.These cases can be regarded as concept proofs indicating targeted engineering might help to create high-efficiency cell factories based upon constructed biological components.