The absence of UDP-GlcNAc in nagA mutant cells translates into the absence of GlcNAc residues in cellulose produced from DnagA cells even under GlcNAc fed conditions. Bacterial cellulose produced by G. xylinus into long, nonaggregated, essentially pure nanofibrils and is a versatile biomaterial due to its unique nanostructure and properties that closely resemble the structure of native extracellular matrices. Despite the excellent biocompatibility and mechanical properties of BC, the lack of cellulose hydrolyzing enzymes in the human body and the high crystallinity restricts its utility. Therefore, cellulose with controllable crystallinity and degradability could be a next generation polymer for tissue engineering applications. Nonetheless the widespread presence of lysozyme in human body warrants its exploitation to degrade a biopolymer containing GlcNAc as one of its constituent. Since the cellulose synthase of G. xylinus can utilize both UDP-glucose and UDP-GlcNAc as substrate further genetic alteration can be carried out in G. xylinus to elevate the UDPGlcNAc pool. This would make UDP-GlcNAc accessible for cellulose synthase and as a result such cells may produce a lysozyme degradable cellulosic heteropolymer consisting of both glucose and GlcNAc. Though we were able to incorporate GlcNAc in cellulose produced from metabolically engineered cells but still we believe that elevated cytoplasmic UDP-GlcNAc could allosterically activate nagA to circumvent the GlcNAc-6-phosphate into other metabolic pathways thereby reducing the level of cytoplasmic UDP-GlcNAc pool. Although disruption of NagA neither induces incorporationof GlcNAc into cellulose nor changes cellulose composition, simultaneous disruption of NagA and heterologous expression of UDP-GlcNac synthesis machinery would likely increase the cytoplasmic UDP-GlcNAc pool. Based on that context, the amount of GlcNac incorporated into cellulose would likely be higher than what was previously achieved. We anticipate that this would lead to the production of a cellulosic heteropolymer consisting of both glucose and GlcNAc as its constituents thereby producing a tailorable chimeric cellulosic biopolymer degradable in human body.
To epigenetic regulation may either too weak to overcome
To estimate the model parameters, data from the metamorphosis assays were converted to percentage metamorphosis success and analyses were conducted using the R statistical platform. In this study we characterised the combined effect of copper and temperature on metamorphosis by fitting Equation 1 to metamorphosis versus Cu concentration data at different temperatures and then analysing how the fitted parameters of the equation changed with temperature. First, we used a nonlinear regression routine to fit Equation 1 to the metamorphosis versus copper data to quantify Mx, EC50 and w at each temperature. Uncertainty in parameter estimates was incorporated using Monte Carlo simulation. To do this we iterated the model 1000 times using parameters randomly drawn from multivariate normal distributions. These distributions were based on the variancecovariance matrices of the parameters describing the relationship between metamorphosis success and Cu concentration at each temperature. In summary, we randomly generated 1000 sets of parameter estimates for each metamorphosis versus Cu relationship, re-fit Equation 2 to each parameter set, and then re-evaluated the fitted model. The simulations were based on uncertainty in the Cu relationship, rather than uncertainty in the parameter temperature relationship, because the former is large relative to the latter. Two approaches were used to determine whether there effects of temperature and Cu concentration were additive, synergistic or antagonistic. First a two-way fixed effects analysis of variance was performed on the larval metamorphosis data. In this analysis, a non-significant interaction term would demonstrate that the effects of temperature and Cu were additive whereas a significant interaction indicates the presence of a synergistic or antagonistic effect. Where an significant interaction was identified, we used an isobologram approach to determine whether the effects were synergistic or antagonistic.