Preliminary Observations on Program Instability

This white paper reports emerging findings at the end of Phase I of the Lean Aircraft Initiative in the Policy focus group area. Specifically, it provides details about research on program instability. Its objective is to discuss high-level findings detailing: 1) the relative contribution of different factors to a program’s overall instability; 2) the cost impact of program instability on acquisition programs; and 3) some strategies recommended by program managers for overcoming and/or mitigating the negative effects of program instability on their programs. Because this report comes as this research is underway, this is not meant to be a definitive document on the subject. Rather, is it anticipated that this research may potentially produce a number of reports on program instability-related topics. The government managers of military acquisition programs rated annual budget or production rate changes, changes in requirements, and technical difficulties as the three top contributors, respectively, to program instability. When asked to partition actual variance in their program’s planned cost and schedule to each of these factors, it was found that the combined effects of unplanned budget and requirement changes accounted for 5.2% annual cost growth and 20% total program schedule slip. At a rate of approximately 5% annual cost growth from these factors, it is easy to see that even conservative estimates of the cost benefits to be gained from acquisition reforms and process improvements can quickly be eclipsed by the added cost associated with program instability. Program management practices involving the integration of stakeholders from throughout the value chain into the decision making process were rated the most effective at avoiding program instability. The use of advanced information technologies was rated the most effective at mitigating the negative impact of program instability.

Retargeting of pre-set regions on chromosome for high gene expression in mammalian cells

We have developed a system to hunt and reuse special gene integration sites that allow for high and stable gene expression. A vector, named pRGFP8, was constructed. The plasmid pRGFP8 contains a reporter gene, gfp2 and two extraneous DNA fragments. The gene gfp2 makes it possible to screen the high expression regions on the chromosome. The extraneous DNA fragments can help to create the unique loci on the chromosome and increase the gene targeting frequency by increasing the homology. After transfection into Chinese hamster ovary cells (CHO) cells, the linearized pRGFP8 can integrate into the chromosome of the host cells and form the unique sites. With FACS, 90 millions transfected cells were sorted and the cells with strongest GFP expression were isolated, and then 8 stable high expression GFP CHO cell lines were selected as candidates for the new host cell. Taking the unique site created by pRGFP8 on the chromosome in the new host cells as a targeting locus, the gfp2 gene was replaced with the gene of interest, human ifngamma, by transfecting the targeting plasmid pRIH-IFN. Then using FACS, the cells with the dimmest GFP fluorescence were selected. These cells showed they had strong abilities to produce the protein of interest, IFN-gamma. During the gene targeting experiment, we found there is positive correlation between the fluorescence density of the GFP CHO host cells and the specific production rate of IFN-gamma. This result shows that the strategy in our expression system is correct: the production of the interesting protein increases with the increase fluorescence of the GFP host cells. This system, the new host cell lines and the targeting vector, can be utilized for highly expressing the gene of interest. More importantly, by using FACS, we can fully screen all the transfected cells, which can reduce the chances of losing the best cells.

Preliminary Observations on Program Instability

This white paper reports emerging findings at the end of Phase I of the Lean Aircraft Initiative in the Policy focus group area. Specifically, it provides details about research on program instability. Its objective is to discuss high-level findings detailing: 1) the relative contribution of different factors to a program’s overall instability; 2) the cost impact of program instability on acquisition programs; and 3) some strategies recommended by program managers for overcoming and/or mitigating the negative effects of program instability on their programs. Because this report comes as this research is underway, this is not meant to be a definitive document on the subject. Rather, is it anticipated that this research may potentially produce a number of reports on program instability-related topics. The government managers of military acquisition programs rated annual budget or production rate changes, changes in requirements, and technical difficulties as the three top contributors, respectively, to program instability. When asked to partition actual variance in their program’s planned cost and schedule to each of these factors, it was found that the combined effects of unplanned budget and requirement changes accounted for 5.2% annual cost growth and 20% total program schedule slip. At a rate of approximately 5% annual cost growth from these factors, it is easy to see that even conservative estimates of the cost benefits to be gained from acquisition reforms and process improvements can quickly be eclipsed by the added cost associated with program instability. Program management practices involving the integration of stakeholders from throughout the value chain into the decision making process were rated the most effective at avoiding program instability. The use of advanced information technologies was rated the most effective at mitigating the negative impact of program instability.

Inverse Metabolic Engineering of Synechocystis PCC 6803 for Improved Growth Rate and Poly-3-hydroxybutyrate Production

Synechocystis PCC 6803 is a photosynthetic bacterium that has the potential to make bioproducts from carbon dioxide and light. Biochemical production from photosynthetic organisms is attractive because it replaces the typical bioprocessing steps of crop growth, milling, and fermentation, with a one-step photosynthetic process. However, low yields and slow growth rates limit the economic potential of such endeavors. Rational metabolic engineering methods are hindered by limited cellular knowledge and inadequate models of Synechocystis. Instead, inverse metabolic engineering, a scheme based on combinatorial gene searches which does not require detailed cellular models, but can exploit sequence data and existing molecular biological techniques, was used to find genes that (1) improve the production of the biopolymer poly-3-hydroxybutyrate (PHB) and (2) increase the growth rate. A fluorescence activated cell sorting assay was developed to screen for high PHB producing clones. Separately, serial sub-culturing was used to select clones that improve growth rate. Novel gene knock-outs were identified that increase PHB production and others that increase the specific growth rate. These improvements make this system more attractive for industrial use and demonstrate the power of inverse metabolic engineering to identify novel phenotype-associated genes in poorly understood systems.