Hurtig Verifikation af Terminators Brug af PGR-Blå plasmid og 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 be completed in a relatively short time frame.
The PGR-Blue plasmid is a modification of the PGR plasmid that has been redesigned to use Golden Gate Assembly (GGA) to simplify the cloning procedure by allowing for all reaction steps to be simultaneously performed in one micro-centrifuge tube.2,3 Color selection was incorporated into the plasmid to increase the ease of identifying positive colonies. A successful ligation should be white/yellow in visible light and fluoresce green under blue (450 nm) or ultraviolet (UV) light when grown on plates containing arabinose. Because uncut pGR-blue contains a blue chromo protein (amilCP), colonies containing an unmodified plasmid are blue under visible light. This simplification along with the streamlined protocol allows researchers to proceed from bioinformatic identification to biological confirmation in three to four days. The design nature of this system can be beneficial both in the research lab and in educational settings.
The pGR-Blue plasmid allows for quantification of terminator strength.4 A single arabinose inducible promoter is used to produce green fluorescent protein (GFP) and red fluorescent protein (RFP). The terminator is cloned into the plasmid after the GFP sequence but before the RFP sequence, thus stopping the transcription of the RFP protein. The strength of the terminator is determined by the ratio of GFP produced to RFP produced.
The Vision and Change5 report suggested that Science, Technology, Engineering and Math (STEM) education incorporate research based experiences into the classroom.6 However, this requires the development of protocols that can be done by students with limited skill sets in a defined time frame. While the protocol can be accomplished in as little as three days, it was also designed so that each major step could be accomplished in a separate weekly (2-3 hr) lab period to create a Course Research Experience (CRE). When used in this manner, the procedure will take between three and six weeks and is appropriate for both introductory and advanced courses in Genetics, Cell Biology or Bioinformatics.
Protocol
Representative Results
Discussion
Det vigtigste skridt i denne protokol er korrekt oligonukleotid design forud for bestilling. Oligonukleotiderne skal have de relevante klæbrige ender tilsat til 5 'enderne af både de øverste og nederste strenge til at sikre, at GGA inkorporering er mulig. Desuden er det vigtigt at skifte orienteringen af venstre står terminatorer (terminatorer, der stopper transkription på nederste streng) til den i korrekt vendende (terminerer transkription på øverste streng) terminatorer fordi GFP og RFP udtryk er i k…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfattere vil gerne anerkende Malcom Campbell og Todd Eckdahl med Genome Consortium for aktiv Undervisning (GCAT) og HHMI-Science Education Alliance – Fag Hunters Advancing Genomforskning og Evolutionary Science (SEA-fager) program.
Dette projekt blev støttet af tilskud fra National Center for Research Resources (P20RR016460) og Statens Institut for General Medical Sciences (P20GM103429) fra National Institutes of Health. Denne forskning blev delvist understøttet af National Science Foundation under tilskud # IIA-1.457.888. Derudover institutionelle (Ouachita Baptist University) midler blev tilvejebragt gennem JD Patterson Summer Research Fellowship.
Materials
| 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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