CO₂ Absorption and Sequestration as Various Polymorphs of CaCO₃ Using Sterically Hindered Amine

One aspect of the attempt to restrain global warming is the reduction of the levels of atmospheric CO₂ produced by fossil fuel power systems. This study attempted to develop a method that reduces CO₂ emissions by investigating the absorption of CO₂ into sterically hindered amine 2-amino-2-methyl-1-propanol (AMP), the acceleration of the absorption rate by using the enzyme carbonic anhydrase (CA), and the conversion of the absorption product to stable carbonates. CO₂ absorbed by AMP is converted via a zwitterion mechanism to bicarbonate species; the presence of these anions was confirmed with ¹H and ¹³C NMR spectral analysis. The catalytic efficiency (<i>k</i>_cat/<i>K</i>_m), CO₂ absorption capacities, and enthalpy changes (Δ<i>H</i>_abs) of aqueous AMP in the presence or absence of CA were found to be 2.61 × 10⁶ or 1.35 × 10² M^–1 s^–1, 0.97 or 0.96 mol/mol, and −69 or −67 kJ/mol, respectively. The carbonation of AMP-absorbed CO₂ was performed by using various Ca²⁺ sources, viz., CaCl₂ (CAC), Ca­(OOCCH₃)₂ (CAA), and Ca­(OOCCH₂CH₃)₂ (CAP), to obtain various polymorphs of CaCO₃. The yields of CaCO₃ from the Ca²⁺ sources were found in the order CAP > CAA > CAC as a result of the effects of the corresponding anions. CAC produces pure rhombohedral calcite, and CAA and CAP produce the unusual phase transformation of calcite to spherical vaterite crystals. Thus, AMP in combination with CAA and CAP can be used as a CO₂ absorbent and buffering agent for the sequestration of CO₂ in porous CaCO₃

Tunable Control of an <i>Escherichia coli</i> Expression System for the Overproduction of Membrane Proteins by Titrated Expression of a Mutant <i>lac</i> Repressor

Most inducible expression systems suffer from growth defects, leaky basal induction, and inhomogeneous expression levels within a host cell population. These difficulties are most prominent with the overproduction of membrane proteins that are toxic to host cells. Here, we developed an <i>Escherichia coli</i> inducible expression system for membrane protein production based on titrated expression of a mutant <i>lac</i> repressor (mLacI). Performance of the mLacI inducible system was evaluated in conjunction with commonly used <i>lac</i> operator-based expression vectors using a T7 or <i>tac</i> promoter. Remarkably, expression of a target gene can be titrated by the dose-dependent addition of l-rhamnose, and the expression levels were homogeneous in the cell population. The developed system was successfully applied to overexpress three membrane proteins that were otherwise difficult to produce in <i>E. coli</i>. This gene expression control system can be easily applied to a broad range of existing protein expression systems and should be useful in constructing genetic circuits that require precise output signals

Charting Microbial Phenotypes in Multiplex Nanoliter Batch Bioreactors

High-throughput growth phenotyping is receiving great attention for establishing the genotype–phenotype map of sequenced organisms owing to the ready availability of complete genome sequences. To date, microbial growth phenotypes have been investigated mostly by the conventional method of batch cultivation using test tubes, Erlenmeyer flasks, or the recently available microwell plates. However, the current batch cultivation methods are time- and labor-intensive and often fail to consider sophisticated environmental changes. The implementation of batch cultures at the nanoliter scale has been difficult because of the quick evaporation of the culture medium inside the reactors. Here, we report a microfluidic system that allows independent cell cultures in evaporation-free multiplex nanoliter reactors under different culture conditions to assess the behavior of cells. The design allows three experimental replicates for each of eight culture environments in a single run. We demonstrate the versatility of the device by performing growth curve experiments with <i>Escherichia coli</i> and microbiological assays of antibiotics against the opportunistic pathogen <i>Pseudomonas aeruginosa</i>. Our study highlights that the microfluidic system can effectively replace the traditional batch culture methods with nanoliter volumes of bacterial cultivations, and it may be therefore promising for high-throughput growth phenotyping as well as for single-cell analyses