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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly

Published: April 25th, 2016

DOI:

10.3791/54064

1Department of Biology, Ouachita Baptist University

This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.

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.

Large synthetic biology projects necessitate the use of highly effective transcription terminators to help regulate gene expression. Identification of novel terminators requires bioinformatic analysis of novel genomes. However, as increasing amounts of bioinformatic software are developed, each with a unique algorithm utilized for prediction, more discrepancy between putative results occurs. Because this process is somewhat subjective and is done in silico, these predictions need biological confirmation.1 Additionally, the volume of putative terminators identified through in-silico analysis requires the use of cloning strategies that can b....

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1. Designing and Ordering Oligonucleotides with the Appropriate Sticky Ends

  1. Identify potential rho-independent terminators through genomic analysis using programs that are freely available online.7
  2. When working with double stranded DNA, determine the orientation of the terminator to be tested.7 The pGR-Blue plasmid only verifies terminators ligated in the 5' to 3' direction on the top (forward strand).
    1. Convert a bottom (reverse) strand terminator to its reve.......

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This protocol will produce cells containing pGR-Blue with a terminator ligated between GFP and RFP using Golden Gate Assembly (Figure 2). Positive colonies containing ligated insert can be selected based on color. In visible light positive colonies will be white/yellow and false positives will produce blue colonies after 18-20 hr of incubation at 37 °C (Figure 3).

After colony selection, a .......

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The most important step in this protocol is proper oligonucleotide design prior to ordering. The oligonucleotides must have the appropriate sticky ends added to the 5' ends of both the top and bottom strands to ensure that GGA incorporation is possible. Additionally, it is important to switch the orientation of left facing terminators (terminators that stop transcription on the bottom strand) to that of right facing (terminates transcription on the top strand) terminators because GFP and RFP expression is in the righ.......

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Authors would like to acknowledge Malcom Campbell and Todd Eckdahl with the Genome Consortium for Active Teaching (GCAT) and the HHMI-Science Education Alliance - Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program.

This project was supported by grants from the National Center for Research Resources (P20RR016460) and the National Institute of General Medical Sciences (P20GM103429) from the National Institutes of Health. This research was supported in part by the National Science Foundation under grant# IIA-1457888. Additionally institutional (Ouachita Baptist University) funds wer....

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Name Company Catalog Number Comments
pGR-Blue Plasmid Addgene 68374
pGR-Plasmid Addgene 46002
AeraSeal-(Sterile Sheets) Excel Scientific BS-25 Sterile Sheets only
10X T4 DNA ligase Buffer NEB
BsaI-HF NEB R3535S The non-HF enzyme will work but is less heat stable. 
NEB Golden Gate Assembly Mix NEB E1600S Commerial Master Mix refered to in the protocol.
T4 DNA ligase NEB M0202S
Round Microcentrifuge Floating Rack Nova Tech International F18875-6401
Ampicillin sodium salt Sigma Aldrich A9518
L-(+)-Arabinose Sigma Aldrich A-3256 D-Arabinose will not induce the pBAD promoter
Luria Base (LB) - Broth, Miller Sigma Aldrich L1900
Luria Base (LB) - Agar , Miller  Sigma Aldrich L2025
Tecan-Infinite M200 Plate Reader Tecan
Mix & Go Competent Cells - Strain JM109 Zymo Research T3005 Use company recommended transformation protocol
ApE: A plasmid editor-software http://biologylabs.utah.edu/jorgensen/wayned/ape/
Tris-HCl, Molecular Grade Promega H5121
Sodium Chloride (Crystalline/Biological, Certified) Fisher Chemical S671
Comercial Oligonucleotide synthesis Integrated DNA Technologies (IDT) http://www.idtdna.com/site
Microtest Tissue Culture Plates- 96 well (Sterile) Falcon 35-3072
mycobacteriophage "Bernal13" Genebank KJ510413
Nuclease Free Water Integrated DNA Technologies (IDT) IDT004
Sterile, L-shaped Hockey-Stick Cell Life Science Products 6444-S1
Nano-Drop 2000c UV-Vis Spectrometer Thermo Scientific 2000c
ARNold: a web tool for the prediction of Rho-independent transcription terminators. http://rna.igmors.u-psud.fr/

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