SDS-PAGE analysis of expression of <i>mmsB</i>, <i>PSEEN0851</i>, and <i>tsf</i> in <i>E. coli</i> JM109.

<p>The transformants were grown in LB at 37°C and induced by 1 mM IPTG. Lanes: M, molecular mass marker; 1, pQE-80L (as control); 2, pQE-<i>mmsB</i>; 3, pQE-<i>PSEEN0851</i>; 4, pQE-<i>tsf</i>. Arrowheads indicate the locations of recombinant proteins.</p

2-DE images of protein extracts of <i>P. putida</i> JUCT1 grown under different solvent conditions.

<p>a: nutrient medium without solvent; b: with 60% (v/v) cyclohexane; c: magnification of c1–c3, the protein spots selected in this study are circled. Arrowheads indicate the protein spots exhibiting intensity discrepancy of over 50% in samples a and b.</p

Primers for PCR amplification of genes <i>mmsB</i>, <i>tsf, and PSEEN0851</i>.

a<p>The underlined sequences are the restriction enzymes sites.</p

Colony formation of <i>E. coli</i> JM109 strains over-expressing <i>mmsB</i>, <i>tsf</i>, and <i>PSEEN0851</i> on LBGMg agar overlaid with decalin after 24 h incubation at 37°C. JM109 carrying empty pQE-80L was used as the control.

<p>Colony formation of <i>E. coli</i> JM109 strains over-expressing <i>mmsB</i>, <i>tsf</i>, and <i>PSEEN0851</i> on LBGMg agar overlaid with decalin after 24 h incubation at 37°C. JM109 carrying empty pQE-80L was used as the control.</p

The oxidation of cyclohexane by <i>E. coli</i> strains.

<p>Incubation condition: 37°C for 8 h with cyclohexane (1%, w/v).</p

MALDI-TOF/TOF analysis of peptides from 5 high-abundance proteins.

a<p>Number of peptides identified for individual protein in MALDI-TOF/TOF analysis.</p>b<p>Number of unique peptides identified for individual protein.</p>c<p>Total amino acid sequence of peptides identified for individual protein.</p>d<p>Percentage of amino acid sequence covered by peptides of individual protein.</p

Five high-abundance protein spots identified by MALDI-TOF/TOF.

a<p>The expression level of <i>P. putida</i> JUCT1 grown in nutrient medium without solvent.</p>b<p>The expression level of <i>P. putida</i> JUCT1 grown in the presence of 60% (v/v) cyclohexane.</p

Effect of organic solvents on cell growth of <i>P. putida</i> JUCT1 (open bar) and its parent strain JUCS (gray bar).

<p>The strains were initially grown in nutrient medium at 37°C till OD₆₆₀ reached 0.2, and then 60% (v/v) organic solvent was added for further incubation of 5 h.</p

Cell growth of recombinant <i>E. coli</i> JM109 strains in the presence of 4% (v/v) cyclohexane.

<p>The recombinant strains were cultured at 37°C and cyclohexane (CH) was added when OD₆₆₀ reached 0.2 (indicated by the arrow). □, pQE-80L (as the control); ▾, pQE-<i>mmsB</i>; •, pQE- <i>tsf</i>; △, pQE-<i>PSEEN0851</i>.</p

Microzone Melting Method of Porous Reactor Fabrication with Structure-Controlled Microchannel Networks for High Yield In Situ DNA Synthesis

This paper presents a simple and cost-effective method for fabricating porous polydimethylsiloxane (PDMS) reactor array chip that is applied in de novo DNA synthesis. A microzone melting technique is proposed in the preparation of a porous PDMS reactor using the sugar particle as a sacrificial template. The curing temperature of 155 °C, higher than the melting point of the sugar particle, is chosen to enhance interconnectivity and reduce internal surface roughness of micropores inside the porous PDMS. The morphological observation and flow resistance test were performed on porous PDMS fabricated with various sugar particle sizes and weight ratios of PDMS to the sugar particle. The results indicate that region I (interconnected pore area) plays a pivotal role in the flow resistance of the porous PDMS reactor. The effectiveness of the porous PDMS reactor in DNA synthesis is verified by gel electrophoresis and fluorescence hybridization. Synthesis product analysis demonstrates that the yield of the porous PDMS reactor is in the same order of magnitude as that of a commercially available 200 nmol synthesis column. The proposed porous PDMS microreactor array chip exhibits great potential in the high-yield DNA synthesis