Interlaboratory Reproducibility in Growth and Reporter Expression in the Cyanobacterium Synechocystis sp. PCC 6803.
Interlaboratory Reproducibility in Growth and Reporter Expression in the Cyanobacterium Synechocystis sp. PCC 6803.
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DOI：
10.1021/acssynbio.3c00150
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发表时间：
2023-06-16
影响因子：
4.7
通讯作者：
Schmelling, Nicolas M.
中科院分区：
文献类型：
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作者：
Mager, Maurice;Hernandez, Hugo Pineda;Brandenburg, Fabian;Lopez-Maury, Luis;McCormick, Alistair J.;Nuernberg, Dennis J.;Orthwein, Tim;Russo, David A.;Victoria, Angelo Joshua;Wang, Xiaoran;Zedler, Julie A. Z.;dos Santos, Filipe Branco;Schmelling, Nicolas M.
近年来，已经发表了大量用于蓝细菌的新合成生物学工具；然而，它们报道的特征往往无法重现，极大地限制了结果的可比性并阻碍了它们的适用性。在这项实验室间研究中，蓝藻模型生物集胞藻的标准微生物实验的可重复性。对 PCC 6803 进行了评估。来自八个不同实验室的参与者量化了 mVENUS 的荧光强度，作为 PJ23100、PrhaBAD 和 PpetE 三个启动子随时间的转录活性的代理。此外，还测量了生长速率以比较实验室之间的生长条件。通过建立严格和标准化的实验室协议，反映经常报告的方法，我们的目的是识别最先进的程序的问题并评估其对再现性的影响。发现不同实验室的相同样品的分光光度计测量值存在显着差异，这表明常用的光密度值报告实践需要通过细胞计数或生物量测量来补充。此外，尽管培养箱中的光强度标准化，但观察到本研究中使用的培养箱之间的生长率存在显着差异，这突出表明除了光强度和二氧化碳供应之外，还需要对光养生物的生长条件进行额外的报告要求。尽管使用了与集胞藻正交的调节系统。 PCC 6803、PrhaBAD 和高水平的方案标准化，在各个实验室中发现诱导条件下启动子活性有约 32% 的变化，这表明蓝藻领域其他数据的可重复性可能会受到类似的影响。
In recent years, a plethora of new synthetic biology tools for use in cyanobacteria have been published; however, their reported characterizations often cannot be reproduced, greatly limiting the comparability of results and hindering their applicability. In this interlaboratory study, the reproducibility of a standard microbiological experiment for the cyanobacterial model organism Synechocystis sp. PCC 6803 was assessed. Participants from eight different laboratories quantified the fluorescence intensity of mVENUS as a proxy for the transcription activity of the three promoters PJ23100, PrhaBAD, and PpetE over time. In addition, growth rates were measured to compare growth conditions between laboratories. By establishing strict and standardized laboratory protocols, reflecting frequently reported methods, we aimed to identify issues with state-of-the-art procedures and assess their effect on reproducibility. Significant differences in spectrophotometer measurements across laboratories from identical samples were found, suggesting that commonly used reporting practices of optical density values need to be supplemented by cell count or biomass measurements. Further, despite standardized light intensity in the incubators, significantly different growth rates between incubators used in this study were observed, highlighting the need for additional reporting requirements of growth conditions for phototrophic organisms beyond the light intensity and CO2 supply. Despite the use of a regulatory system orthogonal to Synechocystis sp. PCC 6803, PrhaBAD, and a high level of protocol standardization, ∼32% variation in promoter activity under induced conditions was found across laboratories, suggesting that the reproducibility of other data in the field of cyanobacteria might be affected similarly.
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影响因子：
3.7
作者：
Beal J;Haddock-Angelli T;Baldwin G;Gershater M;Dwijayanti A;Storch M;de Mora K;Lizarazo M;Rettberg R;with the iGEM Interlab Study Contributors
通讯作者：
with the iGEM Interlab Study Contributors
影响因子：
--
作者：
Myers JA;Curtis BS;Curtis WR
通讯作者：
Curtis WR
影响因子：
6.4
作者：
El-Shehawy, RM;Kleiner, D
通讯作者：
Kleiner, D
影响因子：
4.7
作者：
Liu, Deng;Johnson, Virginia M.;Pakrasi, Himadri B.
通讯作者：
Pakrasi, Himadri B.
影响因子：
5.6
作者：
EGAN, SM;SCHLEIF, RF
通讯作者：
SCHLEIF, RF