Sizeable differences in gene expression means and ratios—based solely upon where genes are located on a plasmid (gene syntax)—have been discovered by researchers at Dartmouth College. This new understanding will help biomanufacturers to design more effective plasmids and genetic circuits and thereby enable more precise measurements of promoter activity, more accurate gene expression, and more predictable engineered systems.
In a recent paper, lead author Yijie Deng, PhD, research scientist, reports with colleagues that, “Genes aligned in the same direction as a plasmid’s origin of replication (Ori) typically exhibit higher expression levels,” than genes placed in the opposite direction. Two adjacent genes in the divergent orientation, they say, “tend to suppress each other’s expression.” Altering gene order while maintaining their orientation yields varied expression levels.
As Deng tells GEN, “To increase protein production, targeted genes should be placed in the same direction as the Ori on a plasmid, which enhances expression. In metabolic engineering, arranging rate-limiting enzyme genes in the right order and orientation can help cells produce more of a desired substance.” Also, he adds, “Avoid placing genes too close together in a divergent orientation, as they can interfere with each other and reduce each other’s expression.”
Simply changing the direction of the green fluorescent protein (GFP) reporter gene on a plasmid lowered its expression level by 12% to 30%, depending on plasmid constructs, Deng and colleagues found. Exchanging positions of the GFP and red fluorescent protein (RFP) altered GFP expression by 15% to 31%, and RPF expression by 4% to 17%.
Gene syntax also affects the behavior of genetic circuits, such as incoherent feedforward loops (iFFLs), and “contributes to unpredictable outcomes in genetic networks,” they found.
In the iFFL version in which the orientation of the transcription factor AraC-RFP and the Ori of the pSC101 plasmid matched, GFP expression peaked at about 70,000 arbitrary units (a.u.) after eight hours, and then reached a steady state. The version using head-on placement never exceeded 10,000 a.u.
That trend carried through to steady-state GFP expression when arabinose was introduced, and to cell-to-cell variation of GFP expression. In each experiment, levels were markedly higher for the iFFLs when the transcription factor and plasmid were codirectional.
Deng says this work also applies to “synthetic biology and smart medicine… such as using engineered microbes to produce insulin in response to blood sugar levels. Achieving such precise regulation depends on careful genetic circuit design—including plasmid architecture—to ensure that the right genes are activated at the right time, and in the right conditions.”
For biomanufacturers, this means greater attention to gene syntax is warranted.
The post Gene Syntax Guidelines to Increase Expression and Predictability appeared first on GEN - Genetic Engineering and Biotechnology News.
In a recent paper, lead author Yijie Deng, PhD, research scientist, reports with colleagues that, “Genes aligned in the same direction as a plasmid’s origin of replication (Ori) typically exhibit higher expression levels,” than genes placed in the opposite direction. Two adjacent genes in the divergent orientation, they say, “tend to suppress each other’s expression.” Altering gene order while maintaining their orientation yields varied expression levels.
As Deng tells GEN, “To increase protein production, targeted genes should be placed in the same direction as the Ori on a plasmid, which enhances expression. In metabolic engineering, arranging rate-limiting enzyme genes in the right order and orientation can help cells produce more of a desired substance.” Also, he adds, “Avoid placing genes too close together in a divergent orientation, as they can interfere with each other and reduce each other’s expression.”
Simply changing the direction of the green fluorescent protein (GFP) reporter gene on a plasmid lowered its expression level by 12% to 30%, depending on plasmid constructs, Deng and colleagues found. Exchanging positions of the GFP and red fluorescent protein (RFP) altered GFP expression by 15% to 31%, and RPF expression by 4% to 17%.
iFFLs also affected
Gene syntax also affects the behavior of genetic circuits, such as incoherent feedforward loops (iFFLs), and “contributes to unpredictable outcomes in genetic networks,” they found.
In the iFFL version in which the orientation of the transcription factor AraC-RFP and the Ori of the pSC101 plasmid matched, GFP expression peaked at about 70,000 arbitrary units (a.u.) after eight hours, and then reached a steady state. The version using head-on placement never exceeded 10,000 a.u.
That trend carried through to steady-state GFP expression when arabinose was introduced, and to cell-to-cell variation of GFP expression. In each experiment, levels were markedly higher for the iFFLs when the transcription factor and plasmid were codirectional.
Deng says this work also applies to “synthetic biology and smart medicine… such as using engineered microbes to produce insulin in response to blood sugar levels. Achieving such precise regulation depends on careful genetic circuit design—including plasmid architecture—to ensure that the right genes are activated at the right time, and in the right conditions.”
For biomanufacturers, this means greater attention to gene syntax is warranted.
The post Gene Syntax Guidelines to Increase Expression and Predictability appeared first on GEN - Genetic Engineering and Biotechnology News.