Name: rapid verification of terminators using the pgr-blue plasmid and golden gate assembly
Uploaded: 2016-04-25T15:00:00
Description: Watch this Scientific Journal Video about Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly at JoVE.com

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 were provided through the J.D. Patterson Summer Research Fellowship.

1. Designing and Ordering Oligonucleotides with the Appropriate Sticky Ends
<ol>
	<li>Identify potential rho-independent terminators through genomic analysis using programs that are freely available online.7</li>
	<li>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&#39; to 3&#39; direction on the top (forward strand).
	<ol>
		<li>Convert a bottom (reverse) strand terminator to its reve...

<ol>
	<li>Li, J., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+promoter+sequence+and+its+strength+in+gene+expression.">Relationship between promoter sequence and its strength in gene expression.</a> Eur Phys J E Soft Matter. 37 (9), (2014).</li><li>Engler, C., Kandzia, R., Marillonnet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+one+pot,+one+step,+precision+cloning+method+with+high+throughput+capability.">A one pot, one step, precision cloning method with high throughput capability.</a> PLoS One. 3 (11), e3647 (2008).</li><li>Lampropoulos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GreenGate---a+novel,+versatile,+and+efficient+cloning+system+for+plant+transgenesis.">GreenGate---a novel, versatile, and efficient cloning system for plant transgenesis.</a> PLoS One. 8 (12), e83043 (2013).</li><li>Chen, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+582+natural+and+synthetic+terminators+and+quantification+of+their+design+constraints.">Characterization of 582 natural and synthetic terminators and quantification of their design constraints.</a> Nat Methods. 10 (7), 659-664 (2013).</li><li>Woodin, T., Carter, V. C., Fletcher, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vision+and+change+in+biology+undergraduate+education,+a+call+for+action--initial+responses.">Vision and change in biology undergraduate education, a call for action--initial responses.</a> CBE Life Sci Educ. 9 (2), 71-73 (2010).</li><li>Vasaly, H. L., Feser, J., Lettrich, M. D., Correa, K., Denniston, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vision+and+change+in+the+biology+community:+snapshots+of+change.">Vision and change in the biology community: snapshots of change.</a> CBE Life Sci Educ. 13 (1), 16-20 (2014).</li><li>Naville, M., Ghuillot-Gaudeffroy, A., Marchais, A., Gautheret, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARNold:+a+web+tool+for+the+prediction+of+Rho-independent+transcription+terminators.">ARNold: a web tool for the prediction of Rho-independent transcription terminators.</a> RNA Biol. 8 (1), 11-13 (2011).</li><li>Campbell, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pClone:+Synthetic+Biology+Tool+Makes+Promoter+Research+Accessible+to+Beginning+Biology+Students.">pClone: Synthetic Biology Tool Makes Promoter Research Accessible to Beginning Biology Students.</a> CBE Life Sci Educ. 13 (2), 285-296 (2014).</li><li>Rhee, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+the+medium+composition+and+plasmid+combination+on+the+growth+of+recombinant+Escherichia+coli+JM109+and+on+the+production+of+the+fusion+protein+EcoRI::SPA.">Influence of the medium composition and plasmid combination on the growth of recombinant Escherichia coli JM109 and on the production of the fusion protein EcoRI::SPA.</a> J Biotechnol. 55 (2), 69-83 (1997).</li><li>Dirla, S., Chien, J. Y., Schleif, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+mutations+in+the+Escherichia+coli+AraC+protein.">Constitutive mutations in the Escherichia coli AraC protein.</a> J Bacteriol. 191 (8), 2668-2674 (2009).</li><li>Schleif, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+the+L-arabinose+operon+of+Escherichia+coli.">Regulation of the L-arabinose operon of Escherichia coli.</a> Trends Genet. 16 (12), 559-565 (2000).</li><li>Kosuri, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composability+of+regulatory+sequences+controlling+transcription+and+translation+in+Escherichia+coli.">Composability of regulatory sequences controlling transcription and translation in Escherichia coli.</a> Proc Natl Acad Sci U S A. 110 (34), 14024-14029 (2013).</li><li>Sharon, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inferring+gene+regulatory+logic+from+high-throughput+measurements+of+thousands+of+systematically+designed+promoters.">Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters.</a> Nat Biotechnol. 30 (6), 521-530 (2012).</li></ol>

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&#39; 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...

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.

<table border="0" cellpadding="0" cellspacing="0" width="887">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">pGR-Blue Plasmid</td>
			<td style="width:243px;">Addgene</td>
			<td style="width:115px;">68374</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pGR-Plasmid</td>
			<td>Addgene</td>
			<td style="width:115px;">46002</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AeraSeal-(Sterile Sheets)</td>
			<td>Excel Scientific</td>
			<td>BS-25</td>
			<td style="width:235px;">Sterile Sheets only</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10X T4 DNA ligase Buffer</td>
			<td>NEB</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">BsaI-HF</td>
			<td>NEB</td>
			<td>R3535S</td>
			<td style="width:235px;">The non-HF enzyme will work but is less heat stable.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">NEB Golden Gate Assembly Mix</td>
			<td>NEB</td>
			<td>E1600S</td>
			<td style="width:235px;">Commerial Master Mix refered to in the protocol.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA ligase</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Round Microcentrifuge Floating Rack</td>
			<td>Nova Tech International</td>
			<td>F18875-6401</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;">Ampicillin sodium salt</td>
			<td>Sigma Aldrich</td>
			<td>A9518</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">L-(+)-Arabinose</td>
			<td>Sigma Aldrich</td>
			<td>A-3256</td>
			<td style="width:235px;">D-Arabinose will not induce the pBAD promoter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Base (LB) - Broth, Miller</td>
			<td>Sigma Aldrich</td>
			<td>L1900</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luria Base (LB) - Agar , Miller&#160;</td>
			<td>Sigma Aldrich</td>
			<td>L2025</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tecan-Infinite M200 Plate Reader</td>
			<td>Tecan</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mix &#38; Go Competent Cells - Strain JM109</td>
			<td>Zymo Research</td>
			<td>T3005</td>
			<td style="width:235px;">Use company recommended transformation protocol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ApE: A plasmid editor-software</td>
			<td style="width:243px;">http://biologylabs.utah.edu/jorgensen/wayned/ape/</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl, Molecular Grade</td>
			<td>Promega</td>
			<td>H5121</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Sodium Chloride (Crystalline/Biological, Certified)</td>
			<td>Fisher Chemical</td>
			<td>S671</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Comercial Oligonucleotide synthesis</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td style="width:235px;">http://www.idtdna.com/site</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Microtest Tissue Culture Plates- 96 well (Sterile)</td>
			<td>Falcon</td>
			<td>35-3072</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mycobacteriophage &#34;Bernal13&#34;</td>
			<td>Genebank</td>
			<td>KJ510413</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease Free Water</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td>IDT004</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile, L-shaped Hockey-Stick Cell</td>
			<td>Life Science Products</td>
			<td>6444-S1</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nano-Drop 2000c UV-Vis Spectrometer</td>
			<td>Thermo Scientific</td>
			<td>2000c</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">ARNold: a web tool for the prediction of Rho-independent transcription terminators.</td>
			<td style="width:243px;">http://rna.igmors.u-psud.fr/</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
	</tbody>
</table>

rapid verification of terminators using the pgr-blue plasmid and golden gate assembly

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.

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 &#176;C (Figure 3).
After colony selection, a ...

Watch this Scientific Journal Video about Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly at JoVE.com

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

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 ...

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