Rapid monitoring of mRNA levels with a molecular beacon during microbial fermentation.
In the microbial fermentation bioreactor, the processes of mRNA transcription, protein translation, and enzyme-catalyzed biosynthesis remain as "black boxes" of industrial monitoring and process control. Monitoring the kinetics of these "black boxes" is very helpful for optimizing and controlling the microbial fermentation process. This study first applied a molecular beacon (MB) to monitor the changes in the mRNA level of the phzC gene during antibiotic phenazine-1-carboxylic acid fermentation. Seven typical MB hybridization buffers were compared, and the effect of formamide on MBs was also studied. The results showed that rapid monitoring of the mRNA level using MBs was feasible. The optimal hybridization buffer for phzC MB was 100 mM Tris, 1 mM MgCl(2), pH 8.0. The optimal hybridization temperature was 35 degrees C, and formamide proved unsuitable for MB hybridization. The limit of detection of phzC MB was 1.67 nM and MB hybridization was complete by 7 min. Given that the time for RNA extraction is 12 min, it is possible that monitoring of phzC mRNA can be completed in less than 20 min. Since production of most amine acids, organic acids, wines, antibiotics, and proteins relies on microbial fermentation, our method may have some potential for application in these other microbial industries.
Dong
Dexian
D
MOE Key Laboratory of Microbial Metabolism, College of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China. dexian@yahoo.cn
Pang
Yanping
Y
Gao
Qian
Q
Huang
Xianqing
X
Xu
Yuquan
Y
Li
Rongxiu
R
D001483:Base Sequence; D002021:Buffers; D002138:Calibration; D005285:Fermentation / Q000187:drug effects / Q000235:genetics*; D005559:Formamides / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005798:Genes, Bacterial / Q000235:genetics; D015336:Molecular Probe Techniques; D015335:Molecular Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D009693:Nucleic Acid Hybridization / Q000187:drug effects; D011549:Pseudomonas / Q000187:drug effects / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D002021:Buffers; D005559:Formamides; D015335:Molecular Probes; D012333:RNA, Messenger; C031066:formamide