Predictive and interpretive simulation of green fluorescent protein expression in reporter bacteria
Predictive and interpretive simulation of green fluorescent protein expression in reporter bacteria
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DOI：
10.1128/jb.183.23.6752-6762.2001
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发表时间：
2001-12-01
影响因子：
3.2
通讯作者：
Lindow, SE
中科院分区：
文献类型：
--
作者：
Leveau, JHJ;Lindow, SE
我们已经制定了一个数值模型，模拟积累的绿色荧光蛋白（GFP）在细菌细胞中的通用启动子-GFP融合。该模型考虑了启动子的活性、GFP成熟为荧光形式所需的时间、GFP对蛋白水解降解的敏感性以及细菌的生长速率。从该模型中，我们得出了一个简单的公式，启动子活性可以很容易地推断，定量从实际测量的GFP荧光在生长的细菌培养物。为了测试公式的有用性，我们确定了LacI阻遏启动子P-AI/O 4/O3响应于诱导剂IPTG（异丙基-β-D-硫代半乳糖苷）浓度增加的活性，并且能够预测P-A1/O 4/O3内两个操纵子位点中的每一个上的LacI阻遏物之间的协同性。在模型的帮助下，我们还定量了GFP[AAV]、GFP[ASV]和GFP [LVA]的蛋白水解降解，这些是在细菌中稳定性降低的GFP的流行变体。由Michaelis-Menten动力学最好地描述，这些变体降解的速率是驱动其合成的启动子活性的函数：弱启动子产生的GFP荧光比强启动子产生的GFP荧光成比例地少。的程度是依赖于物种：效果是更明显的欧文氏菌比大肠杆菌。这种现象具有重要的意义，从细菌的荧光报告的基础上，这些GFP变体的解释。该模型还预测了生长速率对单个细菌的GFP含量的显著影响，如果不考虑这一点，可能会导致对GFP数据的误解。在实践中，我们的模型将有助于事先测试的不同组合的启动子-gfp融合，最适合的应用程序的一个特定的细菌报告菌株，也为实际的GFP荧光数据的解释，获得与该报告。
We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P-AI/O4/O3 in response to increasing concentrations of the inducer IPTG (isopropyl-beta -D-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P-A1/O4/O3. Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP [LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfp fusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.