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 reversed complement to reorient the sequence for testing in pGR-Blue using free online software, e.g., ApE.</li>
	</ol>
	</li>
	<li>After the orientation is determined to be correct, add the sticky end &#34; 5&#39;-CGAC-3&#39; &#34; to the 5&#39; end of the top strand. Add the sticky end &#34; 5&#39;-CCGC-3&#39; &#34; to the 5&#39; end of the bottom strand. 8 This will ensure that ligated insert will be inserted in the appropriate orientation using GGA.</li>
	<li>Once sequences are determined, order as single stranded oligonucleotides commercially.</li>
</ol>
2. Annealing Oligonucleotides (Applies Only to Freeze-dried DNA)
<ol>
	<li>Re-suspend individual oligonucleotides in nuclease free water to a concentration of 100 &#181;M.</li>
	<li>Make 10x annealing buffer: 1 M NaCl and 100 mM Tris-HCl pH 7.4. Annealing buffer can be stored at 4 &#176;C for several weeks. Once annealing buffer is made and oligonucleotides are re-suspended, begin the annealing8.</li>
	<li>For each terminator to be tested, add 16 &#181;l of ultra-pure water to a 1.5 ml microcentrifuge tube.</li>
	<li>Add 2 &#181;l of 10x annealing buffer to each microcentrifuge tube.</li>
	<li>Add 1 &#181;l of the top strand oligonucleotide (100 &#181;M) from the desired terminator.</li>
	<li>Add 1 &#181;l of the bottom strand oligonucleotide (100 &#181;M) from the desired terminator.</li>
	<li>Create a boiling water bath using a 1,000 ml beaker containing approximately 400 ml of tap water and place on a hot plate.</li>
	<li>Place the microcentrifuge tubes from step 2.6 in a float and boil for 4 min. After 4 min, turn off the heat plate but leave the tube and float in the water bath to slowly cool O/N (18 hr) 8. Store the annealed terminators at -20 &#176;C.</li>
</ol>
3. Golden Gate Assembly (Standard Mix &#8212; Cost Efficient)
<ol>
	<li>Make a 40 nM dilution of the annealed terminators. Add 124 &#181;l of nuclease free water to 1 &#181;l of annealed terminators mix (100 &#181;M).
	<ol>
		<li>Centrifuge samples for 30 sec at 10,000 x g and store on ice.</li>
	</ol>
	</li>
	<li>Into a new tube appropriate for the thermocycler being used, add 6 &#181;l of nuclease free water.</li>
	<li>Add 1 &#181;l of commercial DNA Ligase Buffer [300 mM Tris-HCl (pH 7.7-7.8), 100 mM MgCl2, 100 mM DTT, and 10 mM ATP]. Perform all subsequent steps on ice.</li>
	<li>Add 1 &#181;l of pGR-Blue (35-50 ng/&#181;l) destination plasmid to the microcentrifuge tube.</li>
	<li>Add 1 &#181;l of the 40 nM annealed terminators to the microcentrifuge tube.</li>
	<li>Add 0.5 &#181;l commercial high-fidelity BsaI restriction endonuclease to the microcentrifuge tube.</li>
	<li>Add 0.5 &#181;l of commercial DNA ligase to the microcentrifuge tube.</li>
	<li>Centrifuge the reaction mixture at 10,000 x g for 120 sec. Then place the tube directly into the thermocycler.</li>
	<li>Run the thermocycler program: 20 cycles of 1 min at 37 &#176;C followed by 1 min at 16 &#176;C, followed by 1 cycle of 15 min at 37 &#176;C. Use samples immediately or freeze and store at -20 &#176;C.8</li>
</ol>
4. Golden Gate Assembly (Commercial Master Mix&#160;&#8212; Time Efficient)
<ol>
	<li>Add 124 &#181;l of nuclease free water to 1 &#181;l of the 100 &#181;M annealed terminators created in step 2. This dilutes the terminator DNA concentration to 40 nM. Perform this step in a new, labeled 1.5 ml microcentrifuge tube.</li>
	<li>Into a new tube appropriate for the thermocycler, add 14 &#181;l of nuclease free water. Make sure the new tube is labeled accordingly.</li>
	<li>Add 2 &#181;l of Commercial Master Mix Buffer to the microcentrifuge tube.</li>
	<li>Add 1 &#181;l of pGR-Blue plasmid (35-50 ng/&#181;l) to the microcentrifuge tube.</li>
	<li>Add 2 &#181;l of the 40 nM annealed terminators to the microcentrifuge tube.</li>
	<li>Add 1 &#181;l of Commercial Master Mix to the microcentrifuge tube.</li>
	<li>Centrifuge the 40 nM tube at 10,000 x g for 120 sec. Then place the tube directly into the thermocycler.</li>
	<li>Run the thermocycler program: 1 cycle of 60 min at 37 &#176;C followed by 1 cycle of 15 min at 55 &#176;C. Use samples immediately or freeze and store at -20 &#176;C.</li>
</ol>
5. Transformation and Colony Selection
<ol>
	<li>Prepare Luria Base (LB) agar Petri plates containing 1 mM concentration of ampicillin (Amp) and a 10 mM concentration of arabinose (arb). Prepare at least 10 ml of 1 M L-arabinose stock solution because this stock solution will be used in later steps.</li>
	<li>Use any high-efficient (108 - 109 transformants/&#181;g ) chemically competent E. coli (JM109) cells for transformation.9 For best results, add 3-5 &#181;l of the ligation reaction to 50 &#181;l of competent cells. When using chemical competent cells, follow the transformation protocol specific to the cells being used.</li>
	<li>Spread half (25 &#181;l) of the transformed cells onto pre-warmed LB (Amp/arb) agar plates using a sterile L-shaped &#34;hockey stick&#34; and incubate O/N at 37 &#176;C. Due to ampicillin&#39;s rapid rate of decomposition, do not incubate at 37 &#176;C over 24 hr.</li>
	<li>Perform colony selection based on color by visual inspection. A successful ligation should be white/yellow in visible light and fluoresce green under blue (450 nm) or UV light. An unsuccessful ligation will produce a colony that is blue in color under visible light after 18 - 20 hr.</li>
</ol>
6. Verification and Quantification of Terminator
<ol>
	<li>Prepare 5 ml sterile LB-broth tubes with 1 mM ampicillin and 10 mM arabinose. The number of tubes needed is dependent on the number of terminators being tested plus controls (cells with ampicillin resistance that do not fluoresce and cells containing the uncut pGR-Blue plasmid).</li>
	<li>Under visible light pick 2-3 white/yellow colonies (step 5.4) using a sterile loop. Allow the samples to incubate in broths on a shaker at 37 &#176;C and 160 rpm for 16-18 hr. Do not incubate these samples for more than 24 hr.
	<ol>
		<li>Optionally, use a spectrophotometer to determine optical cell density at 600 nm (OD600) to ensure adequate cell growth. Generally, an OD600 of ~0.8-1.0 is observed after 16-18 hr of growth.</li>
	</ol>
	</li>
	<li>Use a sterile 96-well plate for the remaining steps. Conduct these steps in an aseptic environment and in triplicate. Add 199 &#181;l of sterile LB+ Amp/arabinose broth (1 mM ampicillin/10 mM arabinose) to each well. Include room on the plate for three separate controls.
	<ol>
		<li>Use the following three controls: A well (1) containing LB + Amp/arabinose broth only and a well (2) for untransformed competent cells (blank) to serve as a blank. For quantification (3) of the terminator strength, cells containing uncut original pGR or containing pGR-Blue ligated with a non-terminating sequence should be included (reference control for quantification). See Figure 1 for plate reader layout.</li>
	</ol>
	</li>
	<li>Pipet 1 &#181;l of the cell solution from the O/N broths into individual wells of the 96-well plate.</li>
	<li>Seal plates with a breathable cover and shake for 18-20 hr at 37 &#176;C and 160 rpm.</li>
	<li>Using a plate reader, determine OD600 by reading absorbance at 600 nm. Measure GFP fluorescence as follows: excitation 395 and emission 509. Measure RFP fluorescence as follows: excitation 575 and emission 605. After 18-20 hr, the OD600 should be approximately 0.5.</li>
	<li>To normalize for variation in growth, use the equation: <img alt="Equation 1" src="/files/ftp_upload/54064/54064eq1.jpg" /> for both GFP and RFP.</li>
	<li>After normalization, determine relative terminator strength using the equation&#160; <img alt="Equation 2" src="/files/ftp_upload/54064/54064eq2.jpg" /></li>
</ol>

<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 authors have nothing to disclose.

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 right facing (top strand) direction.
Two separate GGA protocols and buffer mixes can be used to test terminators in pGR-Blue. The standard-economy mix consists of material purchase individually and then mixed in the lab 8. The second protocol uses a commercial pre-made GGA master mix. While both work well; the commercial master mix is more time-efficient but is relatively expensive compared to the standard mix. In an education setting, it may be more economical to make the standard GGA mix in advance and freeze aliquots for individual student use.
As a result of modifying pGR for GGA and adding amilCP in the reverse orientation for color selection, the expression of GFP and RFP is limited in the uncut pGR-Blue plasmid and should not be used as the reference control when determining relative terminator strength. However, all modifications are removed during the cloning process allowing for proper quantification of relative terminator strength. While it is best to use the original unmodified pGR plasmid similar results can be achieved by cloning a sequence known not to be a terminator into pGR-Blue. Both controls showed equal levels of GFP and RFP expression (Figure 5) and, more importantly, had similar GFP/RFP ratios.
For tight regulation of gene expression, the pGR-Blue plasmid constitutively expresses the AraC regulator of the pBAD (arabinose inducible) promoter.10,11 Depending on the cell line used a high arabinose concentration may be required for the expression of GFP and RFP. Good results were achieved using a concentration of 10 mM arabinose in the E. coli cell line JM109. Transformed cells should be either white/yellow or blue under visible light. Under UV light or blue light (450 nm), it is common to only see green fluorescence but not red in positively transformed cells. However, RFP fluorescence was detected using the plate reader.
The terminators used as proof of concept for this protocol were identified in the mycobacteriophage Bernal13. However, pGR-Blue only works in E. coli and it is not a shuttle vector that can be expressed in other species of bacteria. The result is that we do not know exactly how the identified terminators would function in their host environment and is a potential limitation. The expression cassette associated with pGR-Blue is flanked by traditional cloning sites and could be moved to an appropriate shuttle vector. This would allow individual researchers to tailor pGR-Blue to their specific bacterial species, if so desired.
While there are other high-throughput sequence analysis procedures available, most use a forward genetics approach. These approaches use site directed mutagenesis or oligo-synthesis to generate hundreds to thousands of potential sequence combinations.12,13 Once created, the effect of each sequence modification is then cataloged. However, pGR-Blue is designed for a more directed reverse genetics application. When analyzing a novel genome in silico, it is common to observe multiple potential terminators in a given area. pGR-Blue was designed to help researchers quickly refine their in silico predictions by allowing them to test putative sequences in vivo. It should be noted the pGR terminator testing plasmid was used to quantitate terminator strength in 582 sequences4, however pGR uses an restriction-enzyme assembly standard (e.g., basic bio-brick assembly (BBA)), a relatively time consuming cloning process compared to GGA.7 The pGR-Blue plasmid was designed to function similarly to pGR but uses a more rapid cloning procedure and colony color selection. These modifications, along with our simplified protocol, make pGR-Blue adaptable to both education and research oriented labs.

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><tbody><tr><td>pGR-Blue Plasmid</td><td>Addgene</td><td>68374</td><td></td></tr><tr><td>pGR-Plasmid</td><td>Addgene</td><td>46002</td><td></td></tr><tr><td>AeraSeal-(Sterile Sheets)</td><td>Excel Scientific</td><td>BS-25</td><td>Sterile Sheets only</td></tr><tr><td>10x T4 DNA ligase Buffer</td><td>NEB</td><td></td><td></td></tr><tr><td>BsaI-HF</td><td>NEB</td><td>R3535S</td><td>The non-HF enzyme will work but is less heat stable.&amp;nbsp;</td></tr><tr><td>NEB Golden Gate Assembly Mix</td><td>NEB</td><td>E1600S</td><td>Commerial Master Mix refered to in the protocol.</td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>Round Microcentrifuge Floating Rack</td><td>Nova Tech International</td><td>F18875-6401</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma Aldrich</td><td>A9518</td><td></td></tr><tr><td>&lt;em&gt;L&lt;/em&gt;-(+)-Arabinose</td><td>Sigma Aldrich</td><td>A-3256</td><td>D-Arabinose will not induce the pBAD promoter</td></tr><tr><td>Luria Base (LB) - Broth, Miller</td><td>Sigma Aldrich</td><td>L1900</td><td></td></tr><tr><td>Luria Base (LB) - Agar, Miller</td><td>Sigma Aldrich</td><td>L2025</td><td></td></tr><tr><td>Tecan-Infinite M200 Plate Reader</td><td>Tecan</td><td></td><td></td></tr><tr><td>Mix &amp;amp; Go Competent Cells - Strain JM109</td><td>Zymo Research</td><td>T3005</td><td>Use company recommended transformation protocol</td></tr><tr><td>ApE: A plasmid editor-software</td><td>http://biologylabs.utah.edu/jorgensen/wayned/ape/</td><td></td><td></td></tr><tr><td>Tris-HCl, Molecular Grade</td><td>Promega</td><td>H5121</td><td></td></tr><tr><td>Sodium Chloride (Crystalline/Biological, Certified)</td><td>Fisher Chemical</td><td>S671</td><td></td></tr><tr><td>Comercial Oligonucleotide synthesis</td><td>Integrated DNA Technologies (IDT)</td><td></td><td>http://www.idtdna.com/site</td></tr><tr><td>Microtest Tissue Culture Plates - 96 well (Sterile)</td><td>Falcon</td><td>35-3072</td><td></td></tr><tr><td>mycobacteriophage &quot;Bernal13&quot;</td><td>Genebank</td><td>KJ510413</td><td></td></tr><tr><td>Nuclease Free Water</td><td>Integrated DNA Technologies (IDT)</td><td>IDT004</td><td></td></tr><tr><td>Sterile, L-shaped Hockey-Stick Cell</td><td>Life Science Products</td><td>6444-S1</td><td></td></tr><tr><td>Nano-Drop 2000c UV-Vis Spectrometer</td><td>Thermo Scientific</td><td>2000c</td><td></td></tr><tr><td>ARNold: a web tool for the prediction of Rho-independent transcription terminators.</td><td>http://rna.igmors.u-psud.fr/</td><td></td><td></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 plate reader can be used to determine terminator strength. As proof of concept, six putative terminators (T1-6) were bioinformatically identified in the mycobacteriophage Bernal13. The results in Figure 4 show relative terminator strength with cells expressing uncut/unmodified pGR as a control. If a predicted terminator has a GFP/RFP ratio similar to pGR (~0.0) or below 0.1, then the sequence was determined to not be a terminator (T5 and T6). However, numerically each tenth increase represents a fold increase in terminator strength relative to the control. For example, T3 had a high ratio of GFP expression relative to RFP and showed a 5-fold increase (0.5 on graph) in terminator strength as compared to the control and was considered a strong terminator (Figure 4). Data was collected in triplicate and averaged for visual representation.
To determine the best reference control for quantitating terminator strength, we compared raw data using the original pGR plasmid containing no terminator and pGR-Blue cloned with a sequence known not be a terminator. Both controls showed similar levels of GFP and RFP expression (Figure 5) and, more importantly, produced similar GFP/RFP ratios.
<img alt="Figure 1" src="/files/ftp_upload/54064/54064fig1.jpg" /> 
Figure 1. Schematic Detailing Plate Reader Layout. Room should be included on the plate reader for three separate controls (Green). A well (1) containing LB + Amp/arabinose broth only and a well (2) for untransformed competent cells (blank) to serve as a blank. For quantification (3) of the terminator strength, cells containing uncut original pGR or containing pGR-Blue ligated with a non-terminating sequence should be included (reference control for quantification). The remainder of the plate (yellow) can be used to test putative terminators. It is suggested all cells be grown in triplicate and averaged before analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54064/54064fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54064/54064fig2.jpg" /> 
Figure 2. Cloning Site Orientation and Expression of pGR-Blue. Constitutive expression of AmilCP (Blue Chromo Protein) is driven in the opposite (bottom strand) direction of GFP and RFP. When digested with BsaI, AmilCP and the BsaI recognition sites are removed and the terminator to be tested is permanently ligated into the plasmid. The top strand and bottom strand of a terminator with the appropriate sticky ends are shown. The AraC regulator and ampicillin resistance genes are in the plasmid but not shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54064/54064fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54064/54064fig3.jpg" /> 
Figure 3. Colony Selection after Cloning. (A) Representative plate after pGR-Blue / terminator ligation (standard mix) and cell transformation. Blue colonies represent unsuccessful ligations and white/yellow colonies (indicated by arrow) are successful ligations. (B) A zoomed in image of the same plate showing representative colonies after transformation. The background color was digitally altered to further highlight colony color differences. Transformed cells (E.coli-JM109) were plated on LB agar plats containing ampicillin and arabinose. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54064/54064fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54064/54064fig4.jpg" /> 
Figure 4. Quantification of Six Different Terminators in pGR-Blue. Six putative terminators (T1-6) were bioinformatically identified in the mycobacteriophage Bernal13. Results show relative fluorescence. All results were normalized using cells containing uncut/unmodified pGR as a control. T3 was found to be a strong terminator. All results represent individual colonies grown in triplicate and averaged for visual representation. Error bars represent deviations between averaged data points. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54064/54064fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54064/54064fig5.jpg" /> 
Figure 5. GFP and RFP Expression in Controls. Expression of GFP and RFP in uncut/unmodified pGR and two different non-terminator sequences inserted into pGR-Blue. All results represent individual colonies grown in triplicate and averaged for visual representation. Error bars represent deviations between averaged data points. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54064/54064fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</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 ...

rapid-verification-terminators-using-pgr-blue-plasmid-golden-gate

Nanyang Technological University

Research

JoVE Journal

Biology

7.6K Views.  Ouachita Baptist University. This protocol utilizes Golden Gate Assembly and the plasmid pGR-blue to rapidly quantify the strength of terminators found in silico.

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

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Genetics

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here is a powerful experimental approach for analyzing the transcription termination defect under physiological conditions. Like the traditional TRO assay, it relies on the presence of a transcriptionally active polymerase beyond the 3&#8242; end of the gene as an indicator of a transcription termination defect1. It overcomes two major problems encountered with the traditional TRO assay. First, it can detect if the polymerase reading through the termination signal is the one that initiated transcription from the promoter-proximal region, or if it is simply representing a pervasively transcribing polymerase that initiated non-specifically from somewhere in the body or the 3&#8242; end of the gene. Secondly, it can distinguish if the transcriptionally active polymerase signal beyond the terminator region is truly the readthrough sense mRNA transcribing polymerase or a terminator-initiated non-coding anti-sense RNA signal. Briefly, the protocol involves permeabilizing the exponentially growing yeast cells, allowing the transcripts that initiated in vivo to elongate in the presence of the BrUTP nucleotide, purifying BrUTP-labelled RNA by the affinity approach, reverse transcribing the purified nascent RNA and amplifying the cDNA using strand-specific primers flanking the promoter and the terminator regions of the gene2.

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes.

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Biochemistry

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

Bioengineering

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the CRISPR-Cas systems and their natural inhibitors-the anti-CRISPR proteins (Acrs)-provides a new tool to design and implement in vivo gene digital circuits. Here, we describe a protocol that follows the idea of the "Design-Build-Test-Learn" biological engineering cycle and makes use of dCas9/dCas12a together with their corresponding Acrs to establish small transcriptional networks, some of which behave like Boolean gates, in Saccharomyces cerevisiae. These results point out the properties of dCas9/dCas12a as transcription factors. In particular, to achieve maximal activation of gene expression, dSpCas9 needs to interact with an engineered scaffold RNA that collects multiple copies of the VP64 activation domain (AD). In contrast, dCas12a shall be fused, at the C terminus, with the strong VP64-p65-Rta (VPR) AD. Furthermore, the activity of both Cas proteins is not enhanced by increasing the amount of sgRNA/crRNA in the cell. This article also explains how to build Boolean gates based on the CRISPR-dCas-Acr interaction. The AcrIIA4 fused hormone-binding domain of the human estrogen receptor is the core of a NOT gate responsive to &#946;-estradiol, whereas AcrVAs synthesized by the inducible GAL1 promoter permits to mimic both YES and NOT gates with galactose as an input. In the latter circuits, AcrVA5, together with dLbCas12a, showed the best logic behavior.

CRISPR-Cas systems and anti-CRISPR proteins were integrated into the scheme of Boolean gates in Saccharomyces cerevisiae. The new small logic circuits showed good performance and deepened the understanding of both dCas9/dCas12a-based transcription factors and the properties of anti-CRISPR proteins.

Synthetic gene Boolean gates and digital circuits have a broad range of applications, from medical diagnostics to environmental care. The discovery of the ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1&#945;) promoter to drive gene expression. Plasmid vectors containing hEF1&#945; are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1&#945; promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1&#945; promoter part 1, hEF1&#945; promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...

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

Summary

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Chapters in this video

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Mouse Genome Engineering Using Designer Nucleases

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks