The authors have no conflicts of interest to declare.
Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum
Article first published online: 3 JUN 2013
© 2013 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 110, Issue 11, pages 2959–2969, November 2013
How to Cite
Yim, S. S., An, S. J., Kang, M., Lee, J. and Jeong, K. J. (2013), Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum. Biotechnol. Bioeng., 110: 2959–2969. doi: 10.1002/bit.24954
- Issue published online: 21 SEP 2013
- Article first published online: 3 JUN 2013
- Accepted manuscript online: 1 MAY 2013 05:15AM EST
- Manuscript Accepted: 22 APR 2013
- Manuscript Revised: 26 MAR 2013
- Manuscript Received: 9 JAN 2013
- Ministry of Education, Science and Technology of Korea. Grant Number: ABC-2011-0031363
- Brain Korea 21 Project
Additional supporting information may be found in the online version of this article at the publisher's web-site.
Figure S1. The numerical summary of nucleotides at 80 randomized positions. Nucleotide frequency of (a) 40 random clones in original promoter library and (b) 20 clones isolated by FACS screening. Boxed sequences represent two invariable site—RBS “AGGA” and BamHI site (GGATCC). In (b), the conserved sequence (GTAnnaTnG) was shown in red-color letters and underlined.
Figure S2. Mean fluorescence intensities of all 80 isolated clones.
Figure S3. Correlation between GFP expression level (x-axis), mRNA transcript level (y-axis) and mean fluorescence intensity (z-axis). The measured data were shown in Table S3.
Figure S4. Activity of 20 synthetic promoters in E. coli. a: GFP fluorescence intensities of the synthetic promoters in pCES208 derivatives with GFP in E. coli were analyzed by flow cytometry. E. coli (pCES208) was used as negative control. b: The level of GFP expression in each E. coli clone was analyzed by Western blotting. Arrow indicates GFP (∼30 kDa).
Figure S5. Activity of two synthetic promoters (PH30 and PH36) in E. coli under IPTG induction (left) and no induction (right). The fluorescent intensities of the E. coli harboring pET22b (red), pET22b-T7-GFP (cyan), pET22b-H30-GFP (yellow), and pET22b-H36-GFP (green) were analyzed by flow cytometry.
Table SI. List of PCR primers used in this study.
Table S2. Summary of FACS screening of synthetic promoter library.
Table S3. The three different measures (mean fluorescence value by FACS, quantification cycle by qRT-PCR, GFP expression level by Western blotting) from the 20 synthetic promoters and trc promoter.
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