ribosome binding sites (rbs)  (Rocha labs)

Journal: Translational Vision Science & Technology

Article Title: Therapeutic Ultrasound for Topical Corneal Delivery of Macromolecules

doi: 10.1167/tvst.11.8.23

Figure Lengend Snippet: Experimental configuration. ( A ) Diffusion cell schematic. Macromolecule, small ultra-red fluorescent protein (smURFP) is added to the donor compartment ( top ), and Dulbecco's phosphate-buffered saline (DPBS) is added to the receiver compartment ( bottom ). The dissected cornea forms a barrier between the donor and receiver compartments. The transducer is placed in the donor compartment for ultrasound application. Ultrasound is applied to allow macromolecule entry into the corneal epithelium and individual epithelial cells. ( B ) Experimental setup. Three diffusion cells were placed inside a water bath at 34°C with a thermometer also inside the water bath to verify the temperature. The ultrasound transducer ( bottom-left corner ) is shown after it was removed from the donor compartment following the application of ultrasound pulse. ( C ) An example of a custom-designed, circular unfocused 400 kHz ultrasound transducer used in the experiments. The applied ultrasound transducers work at frequencies of 400 or 600 kHz with a 15 mm active diameter.

Article Snippet: Briefly, pBAD smURFP – ribosomal binding site (RBS) – Synechocystis heme oxygenase-1 (HO-1) plasmid DNA (#80341; Addgene, Watertown, MA) was transformed in TOP10 E. coli (C404010; Thermo Fisher, Waltham, MA) and grown at 37°C in LB supplemented with 50 μg/mL ampicillin (A0166; Millipore Sigma, Burlington, MA) and 0.2% (w/v) arabinose (100706; MP Biomedicals, Irvine, CA) in the dark.

Techniques: Diffusion-based Assay, Saline

Journal: Translational Vision Science & Technology

Article Title: Therapeutic Ultrasound for Topical Corneal Delivery of Macromolecules

doi: 10.1167/tvst.11.8.23

Figure Lengend Snippet: The schematic outlines the experimental procedure and analysis timeline. Dissected corneas are placed in diffusion cells equilibrated in the water bath. The ultrasound transducers are placed above the cornea in a 7 µM smURFP solution. The sham group receives no ultrasound ( top ), and the ultrasound group receives 400 or 600 kHz ultrasound with an intensity of 1.0 or 0.8 W/cm 2 , respectively, for 5 minutes. The ultrasound transducers are then removed, and the dissected cornea is allowed to incubate in the diffusion cell for 55 minutes, for a total time of 60 minutes. The dissected corneas are collected for histology processing, and the receiver compartment solution is collected and analyzed for spectrophotometry. Corneas are fixed and sectioned. H&E staining of the cornea sections is performed, and the sections are then imaged with a color camera for structure and damage analysis. Fluorescence imaging is performed on unstained cornea sections to confirm macromolecule smURFP entry into the corneal epithelium, and individual epithelial cells.

Article Snippet: Briefly, pBAD smURFP – ribosomal binding site (RBS) – Synechocystis heme oxygenase-1 (HO-1) plasmid DNA (#80341; Addgene, Watertown, MA) was transformed in TOP10 E. coli (C404010; Thermo Fisher, Waltham, MA) and grown at 37°C in LB supplemented with 50 μg/mL ampicillin (A0166; Millipore Sigma, Burlington, MA) and 0.2% (w/v) arabinose (100706; MP Biomedicals, Irvine, CA) in the dark.

Techniques: Diffusion-based Assay, Spectrophotometry, Staining, Fluorescence, Imaging

Journal: Translational Vision Science & Technology

Article Title: Therapeutic Ultrasound for Topical Corneal Delivery of Macromolecules

doi: 10.1167/tvst.11.8.23

Figure Lengend Snippet: Representative images showing the anterior cornea epithelium. Corneas were incubated with 7 µM smURFP and received ( A ) control (sham treatment) or ( B ) ultrasound (600 kHz with an applied intensity of 0.8 W/cm 2 ) for 5 minutes. Brightfield, colored images, and fluorescence, monochrome images are shown on the left and right , respectively. A Control (sham) experiments show dim fluorescence. B Ultrasound delivers smURFP to the interior of the epithelium and inside individual epithelial cells. Control (sham treatment) images A do not show fluorescence inside the epithelium or the cells. The scale bar is 50 µm.

Article Snippet: Briefly, pBAD smURFP – ribosomal binding site (RBS) – Synechocystis heme oxygenase-1 (HO-1) plasmid DNA (#80341; Addgene, Watertown, MA) was transformed in TOP10 E. coli (C404010; Thermo Fisher, Waltham, MA) and grown at 37°C in LB supplemented with 50 μg/mL ampicillin (A0166; Millipore Sigma, Burlington, MA) and 0.2% (w/v) arabinose (100706; MP Biomedicals, Irvine, CA) in the dark.

Techniques: Incubation, Control, Fluorescence

Journal: Translational Vision Science & Technology

Article Title: Therapeutic Ultrasound for Topical Corneal Delivery of Macromolecules

doi: 10.1167/tvst.11.8.23

Figure Lengend Snippet: Representative images of an ultrasound-treated cornea. ( A ) Entire cornea section. Tears result from the sectioning of the cornea dome. The outer cornea is exposed to smURFP and shows the greatest fluorescence. ( B ) Twenty times (20 X) objective image of the red square in A rotated 180 degrees. The epithelium layer shows the fluorescence of the smURFP macromolecule. ( C ) A single image from the red square in B . Single epithelium cells show uptake of smURFP and deeper fluorescence in the stroma. We have entire cornea images for 31 cornea experiments, and additional examples are shown in .

Article Snippet: Briefly, pBAD smURFP – ribosomal binding site (RBS) – Synechocystis heme oxygenase-1 (HO-1) plasmid DNA (#80341; Addgene, Watertown, MA) was transformed in TOP10 E. coli (C404010; Thermo Fisher, Waltham, MA) and grown at 37°C in LB supplemented with 50 μg/mL ampicillin (A0166; Millipore Sigma, Burlington, MA) and 0.2% (w/v) arabinose (100706; MP Biomedicals, Irvine, CA) in the dark.

Techniques: Fluorescence

Journal: Translational Vision Science & Technology

Article Title: Therapeutic Ultrasound for Topical Corneal Delivery of Macromolecules

doi: 10.1167/tvst.11.8.23

Figure Lengend Snippet: Representative images of whole cornea epithelium. Corneas are incubated with 7 µM smURFP received ( A ) control (sham treatment) or ( B ) ultrasound (600 kHz with applied intensity of 0.8 W/cm 2 ) for 5 minutes. Brightfield, colored images, and fluorescence, monochrome images are shown on left and right , respectively. A Control (sham treatment) experiments show weak fluorescence on the exterior of the epithelium or the sclera. B Ultrasound delivers fluorescent proteins to the interior of the epithelium. The anterior face of the epithelium and the sclera are labeled. The cornea epithelium is enclosed in a black dashed box . The red boxes show the locations imaged at higher magnification (20 X), shown in . The scale bar is 2000 µm.

Article Snippet: Briefly, pBAD smURFP – ribosomal binding site (RBS) – Synechocystis heme oxygenase-1 (HO-1) plasmid DNA (#80341; Addgene, Watertown, MA) was transformed in TOP10 E. coli (C404010; Thermo Fisher, Waltham, MA) and grown at 37°C in LB supplemented with 50 μg/mL ampicillin (A0166; Millipore Sigma, Burlington, MA) and 0.2% (w/v) arabinose (100706; MP Biomedicals, Irvine, CA) in the dark.

Techniques: Incubation, Control, Fluorescence, Labeling

Journal: Translational Vision Science & Technology

Article Title: Therapeutic Ultrasound for Topical Corneal Delivery of Macromolecules

doi: 10.1167/tvst.11.8.23

Figure Lengend Snippet: Absorbance of the receiver compartment solution determined at smURFP absorbance maximum of 640 nm. Sham group ( n = 11), 400 kHz ultrasound group ( n = 13), and 600 kHz ultrasound group ( n = 7). The dashed, horizontal lines are the means and the error bars are the standard error of the mean (SEM). Absorbance measurements indicate no statistical difference among the sham and both ultrasound groups.

Article Snippet: Briefly, pBAD smURFP – ribosomal binding site (RBS) – Synechocystis heme oxygenase-1 (HO-1) plasmid DNA (#80341; Addgene, Watertown, MA) was transformed in TOP10 E. coli (C404010; Thermo Fisher, Waltham, MA) and grown at 37°C in LB supplemented with 50 μg/mL ampicillin (A0166; Millipore Sigma, Burlington, MA) and 0.2% (w/v) arabinose (100706; MP Biomedicals, Irvine, CA) in the dark.

Techniques:

Journal: Synthetic and Systems Biotechnology

Article Title: Novel switchable ECF sigma factor transcription system for improving thaxtomin A production in Streptomyces

doi: 10.1016/j.synbio.2022.05.010

Figure Lengend Snippet: The functionality of the heterologous ECF transcriptional system in S. venezuelae ISP5230. A , Genetic layout of the ECF transcription system. (ECF X : ECF11_3726, ECF11_987, ECF16_3622, ECF16_973, ECF34_1384, ECF27_4265, ECF38_1322 (named as ECF38), ECF17_1458 (named as ECF17); the corresponding P ecfx : P ecf11_3726 , P ecf11_987 , P ecf16_3622 , P ecf16_973 , P ecf34_1384 , P ecf27_4265 , P ecf38_1322 (named as P ecf38 ), P ecf17_1458 (named as P ecf17 ). The kasO p* promoter was used as a positive control. The pPAP-P ecfx plasmid in S. venezuelae ISP5230 solely was used as a negative control to exclude potential interference from the host transcription machinery. B , The activities of ECF σ factors in S. venezuelae ISP5230 were monitored by confocal fluorescence microscopy and processed by ImageJ software. C , Precise quantification of P ecf17 promoter activity using flow cytometry. Data represent the mean ± SD of at least three replicate experiments, a.u. means arbitrary unit, ** indicates significant difference.

Article Snippet: Specifically, the kasO p* promoter, rbs-100 ribosome binding site (RBS), codon-optimized ECF σ g e n e s ( T a b l e S 1 ) , a n d t w o f l a n k i n g Bsa I restriction sites were synthesized by the GenScript company, and assembled on the pTHS plasmid via Golden gate method to generate eight pTHS -ECFx plasmids ( ).

Techniques: Positive Control, Plasmid Preparation, Negative Control, Fluorescence, Microscopy, Software, Activity Assay, Flow Cytometry