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

Summary

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

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.

Introduction

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....

Protocol

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 reversed complement to reorient the sequence for testing in pGR-Blue using free online software, e.g., ApE.
3. After the orientation....

Results

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 plate.......

Discussion

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.......

Materials

List of materials used in this article

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/