Department
Biology
Document Type
Article
Publication Date
4-1-2016
Abstract
The goal of this protocol is to allow for the rapid verification of bioinformatically identified terminators. Further, the plasmid (pGR-Blue) is designed specifically for this protocol and allows for the quantification of terminator efficiency. As a proof of concept, six terminators were bioinformatically identified in the mycobacteriophage Bernal13. Once identified, terminators were then made as oligonucleotides with the appropriate sticky ends and annealed together. Using Golden Gate Assembly (GGA), terminators were then cloned into pGR-Blue. Under visible light, false positive colonies appear blue and positively transformed colonies are white/yellow. After induction of an arabinose inducible promoter (pBad) with arabinose, colony strength can be determined by measuring the ratio of green fluorescent protein (GFP) produced to red fluorescent protein (RFP) produced. With pGR-Blue, the protocol can be completed in as little as three days and is ideal in an educational setting. Additionally, results show that this protocol is useful as a means for understanding in silico predictions of terminator efficiency related to the regulation of transcription.
Publication Title
Journal of Visualized Experiments
Publisher Statement
Copyright © 2016 Journal of Visualized Experiments
DOI
10.3791/54064
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Recommended Citation
Bradshaw, J. C., Gongola, A. B., Reyna, N. S. "Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly," Journal of Visualized Experiments (110), e54064, doi:10.3791/54064 (2016).
S1: Materials List
Comments
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