Govind Rao

A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase.

Abstractu2002Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of the gene for green fluorescent protein (GFP) to the 5′ end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP. Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations with inexpensive instrumentation based on detecting green fluorescence.

Low cost phase-modulation measurements of nanosecond fluorescence lifetimes using a lock-in amplifier

The use of a 200 MHz lock-in amplifier was demonstrated as a low cost instrument for frequency domain measurements of nanosecond fluorescence lifetimes. The lock-in directly provided both the dc bias and the ac signal used to modulate the intensity of a blue light emitting diode excitation source. The emission was measured by a photomultiplier tube and the resulting signal was sent back through a dc block to the lock-in with no external signal processing or heterodyning required. The system was highly accurate at measuring phase and modulation up to 80 MHz and moderately so up to 100 MHz. The fluorescence lifetimes of several standard fluorophores (Fluorescein, Rhodamine B, and [Ru(bpy)3]2+) were measured by the lock-in, and the results agreed closely with those made on a research grade fluorometer. The entire lock-in based system costs less than US

Ratiometric oxygen sensing: detection of dual-emission ratio through a single emission filter

10,000 to build and can be controlled by any standard computer through a GPIB or serial connection. The system is also portable, consumes little power, and c...

Generating controlled reducing environments in aerobic recombinant Escherichia coli fermentations: Effects on cell growth, oxygen uptake, heat shock protein expression, and in vivo CAT activity

A new method and device for the ratiometric measurement of oxygen concentration are presented. They are based on the use of a dual-emission oxygen-sensitive dye. The method allows the exclusion of the influence of emission overlap. The detection of the dual-emission ratio is performed using a single long-pass emission filter. The device described is simpler than the widely used lifetime instruments and could easily be a stand-alone low-cost instrument.

Low-cost device for ratiometric fluorescence measurements

The independent control of culture redox potential (CRP) by the regulated addition of a reducing agent, dithiothreitol (DTT) was demonstrated in aerated recombinant Escherichia coli fermentations. Moderate levels of DTT addition resulted in minimal changes to specific oxygen uptake, growth rate, and dissolved oxygen. Excessive levels of DTT addition were toxic to the cells resulting in cessation of growth. Chloramphenicol acetyltransferase (CAT) activity (nmoles/microgram total protein min.) decreased in batch fermentation experiments with respect to increasing levels of DTT addition. To further investigate the mechanisms affecting CAT activity, experiments were performed to assay heat shock protein expression and specific CAT activity (nmoles/microgram CAT min.). Expression of such molecular chaperones as GroEL and DnaK were found to increase after addition of DTT. Additionally, sigma factor 32 (sigma32) and several proteases were seen to increase dramatically during addition of DTT. Specific CAT activity (nmoles/microgram CAT min. ) varied greatly as DTT was added, however, a minimum in activity was found at the highest level of DTT addition in E. coli strains RR1 [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a maximum at the same levels of DTT. Although DTT addition has the potential for directly affecting intracellular protein folding, the effects felt from the increased stress within the cell are likely the dominant effector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also altered. The changes in specific CAT activity, molecular chaperones, and other heat shock proteins, in the presence of minimal growth rate and oxygen uptake alterations, suggest that the ex vivo control of redox potential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations.

Method and apparatus to perform trans-cutaneous analyte monitoring

An all-solid-state, low-cost device for fluorescent wavelength-ratiometric detection is described. Ultrabright light-emitting diodes were used as light sources. This allowed electronic modulation of the light, simple optical configuration, and miniaturization of the instrument. Narrow-bandpass interference filters were used for wavelength separation. Detection was accomplished by high-sensitivity, large-surface PIN photodiodes. An integrating double-ramp technique with fixed upper and lower thresholds was employed for conversion of the light intensities into time intervals. The duty ratio of the output signal was a function of the fluorescence intensity ratio. Additionally, the concentration of the fluorophore could be measured. The device could be easily designed as a battery-operated version. It could be used for a variety of ratiometric fluorescence measurements.