Background
This study compares students' perceptions of key cognitive processes and specific content afforded by an industrially situated virtual laboratory project and two physical laboratory projects.
Purpose(Hypothesis)
1. How do students' perceptions of the nature of cognition, experimental design, and ambiguity compare across selected virtual and physical laboratory experiences? 2. In what ways do students perceive the virtual and physical laboratories as an authentic experience that is reflective of real‐life engineering?
Design/Method
Three, free‐response survey questions were quantitatively and qualitatively analyzed. Content analysis was used to establish categories to group the responses, and the coding process had an interrater reliability of 0.90.
Results
Student responses showed statistically significant increases in categories of Experimental Design, Critical Thinking, and Ambiguity in the virtual laboratories and in Lab Protocol and Specific Content in the physical laboratories. Additionally, more overall High Cognition statements were observed in the virtual laboratory. Student perceptions of ambiguity shifted from ambiguity in the instruction to an ambiguity in the experimental process itself, and many students were able to suspend disbelief in the virtual laboratories and demonstrated psychological presence, leading to the potential for a rich learning experience.
Conclusion
The industrially situated virtual laboratories reported in this paper provide affordances for substantially different student thinking about their thinking than the physical laboratories in the same course. This conclusion does not suggest that the differences are a direct result of the medium of the laboratory (virtual vs. physical) but rather the opportunities that the instructional design of each type of laboratory affords.
A gene encoding manganese peroxidase (mnp1) from Phanerochaete chrysosporium was cloned downstream of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter in the methylotrophic yeast Pichia pastoris. Three different expression vectors were constructed: pZBMNP contains the native P. chrysosporium fungal secretion signal, palphaAMNP contains an alpha-factor secretion signal derived from Saccharomyces cerevisiae, and pZBIMNP has no secretion signal and was used for intracellular expression. Both the native fungal secretion signal sequence and alpha-factor secretion signal sequence directed the secretion of active recombinant manganese peroxidase (rMnP) from P. pastoris transformants. The majority of the rMnP produced by P. pastoris exhibited a molecular mass (55-100 kDa) considerably larger than that of the wild-type manganese peroxidase (wtMnP, 46 kDa). Deletion of the native fungal secretion signal yielded a molecular mass of 39 kDa for intracellular rMnP in P. pastoris. Treatment of the secreted rMnP with endoglycosidase H (Endo H) resulted in a considerable decrease in the mass of rMnP, indicating N-linked hyperglycosylation. Partially purified rMnP showed kinetic characteristics similar to those of wtMnP. Both enzymes also had similar pH stability profiles. Addition of exogenous Mn(II), Ca(II), and Fe(III) conferred additional thermal stability to both enzymes. However, rMnP was slightly less thermostable than wtMnP, which demonstrated an extended half-life at 55 degrees C.
The use of silicon dioxide (SiO(2) ) nanosprings as supports for immobilized enzymes in a continuous microreactor is described. A nanospring mat (2.2 cm(2) × 60 μm thick) was functionalized with γ-aminopropyltriethoxysilane, then treated with N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) and dithiothreitol (DTT) to produce surface thiol (--SH) groups. SPDP-modified β-galactosidase from Aspergillus oryzae was immobilized on the thiolated nanosprings by reversible disulfide linkages. The enzyme-coated nanospring mat was placed into a 175-μm high microchannel, with the mat partially occluding the channel. The kinetics and steady-state conversion of hydrolysis of o-nitrophenyl β-D-galactosylpyranoside at various substrate flow rates and concentrations were measured. Substantial flow was observed through the nanosprings, for which the Darcy permeability κ ≈ 3 × 10(-6) cm(2) . A simple, one-parameter numerical model coupling Navier-Stokes and Darcy flow with a pseudo-first-order reaction was used to fit the experimental data. Simulated reactor performance was sensitive to changes in κ and the height of the nanospring mat. Permeabilities lower than 10(-8) cm(2) practically eliminated convective flow through the nanosprings, and substantially decreased conversion. Increasing the height of the mat increased conversion in simulations, but requires more enzymes and could cause sealing issues if grown above channel walls. Preliminary results indicate that in situ regeneration by reduction with DTT and incubation with SPDP-modified β-galactosidase is possible. Nanosprings provide high solvent-accessible surface area with good permeability and mechanical stability, can be patterned into existing microdevices, and are amenable to immobilization of biomolecules. Nanosprings offer a novel and useful support for enzymatic microreactors, biosensors, and lab-on-chip devices.
A method for the production and concentration of the lignin-degrading enzyme, manganese peroxidase (rMnP), was developed using the yeast Pichia pastoris in high cell density, fed-batch cultivations. A gene encoding manganese peroxidase (mnp1) from the white-rot fungus Phanerochaete chrysosporium was cloned into a protease deficient (pep4-) strain of the methylotrophic yeast P. pastoris. Heme is an important cofactor for active rMnP production, and amendment of yeast cultures with heme increased active rMnP concentrations. In both shake-flasks and fed-batch bioreactors, the relationship between heme concentration and rMnP activity was logarithmic, with increasing heme concentrations resulting in progressively lesser increases in enzyme activity. Scale-up from shake-flasks to 2 L fed-batch cultivations increased rMnP activities from 200 U/L to 2,500 U/L, with addition of 0.1 g/L heme (added heme per liquid volume) at the beginning of the fed-batch phase resulting in higher enzyme activities than addition at the beginning of the batch phase. A combination of centrifugation, acetone precipitation, dialysis, and freeze drying was found to be effective for concentrating the rMnP from 2,500 U/L in the P. pastoris bioreactor culture to 30,000 U/L in 0.1 M potassium phosphate buffer pH 6. The rMnP recovery yield was 60% and the purity was 1-4%. By using 0.1 g/L heme during the fed-batch cultivation, the heme content of the final enzyme preparation could be reduced by 97%, and had sufficiently high rMnP activity and low enough color to be suitable for pulp bleaching experiments.
A tod-lux transcriptional fusion bioluminescent reporter strain, Pseudomonas putida B2, was developed to permit on-line analysis of trichloroethylene (TCE) transformation by toluene dioxygenase (todC1C2BA) in Pseudomonas putida F1. Strain B2 was exposed to toluene in growing and resting cell bioluminescence assays. The growing cells showed a direct correlation between bioluminescence and toluene concentration, while resting cells showed reproducible bioluminescence with repeated toluene exposures. In addition, P. putida B2 was encapsulated in alginate beads and used in a packed bed flow-through differential volume reactor. The TCE feed into the differential volume reactor was constant at 20 mg L-1 and toluene was pulsed in square-wave perturbations at 10 mg L-1. The system showed a direct correlation between the expression of the tod operon (as monitored by light output) and the co-metabolism of TCE. Approximately 20% of the TCE and 50% of the toluene was removed at a flow rate of 0.4 ml min-1. This approach allowed the on-line monitoring of tod gene expression and its relation to TCE biotransformation.
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