Name: Rapid Verification of Terminators Using the pGR-Blue Plasmid and Golden Gate Assembly (Video) | JoVE Journal
Uploaded: 2016-04-25T15:00:00.000+00:00
Duration: 9 min 51 s
Description: Discover rapid terminator verification using Golden Gate Assembly with the pGR-Blue plasmid. This synthetic biology method quantifies terminator strength in vitro within one week.

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5 minutes

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' to 3' 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 " 5'-CGAC-3' " to the 5' end of the top strand. Add the sticky end " 5'-CCGC-3' " to the 5' 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 µM.</li>
	<li>Make 10x annealing buffer: 1 M NaCl and 100 mM Tris-HCl pH 7.4. Annealing buffer can be stored at 4 °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 µl of ultra-pure water to a 1.5 ml microcentrifuge tube.</li>
	<li>Add 2 µl of 10x annealing buffer to each microcentrifuge tube.</li>
	<li>Add 1 µl of the top strand oligonucleotide (100 µM) from the desired terminator.</li>
	<li>Add 1 µl of the bottom strand oligonucleotide (100 µM) 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 °C.</li>
</ol>
3. Golden Gate Assembly (Standard Mix — Cost Efficient)
<ol>
	<li>Make a 40 nM dilution of the annealed terminators. Add 124 µl of nuclease free water to 1 µl of annealed terminators mix (100 µM).
	<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 µl of nuclease free water.</li>
	<li>Add 1 µl 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 µl of pGR-Blue (35-50 ng/µl) destination plasmid to the microcentrifuge tube.</li>
	<li>Add 1 µl of the 40 nM annealed terminators to the microcentrifuge tube.</li>
	<li>Add 0.5 µl commercial high-fidelity BsaI restriction endonuclease to the microcentrifuge tube.</li>
	<li>Add 0.5 µl 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 °C followed by 1 min at 16 °C, followed by 1 cycle of 15 min at 37 °C. Use samples immediately or freeze and store at -20 °C.8</li>
</ol>
4. Golden Gate Assembly (Commercial Master Mix — Time Efficient)
<ol>
	<li>Add 124 µl of nuclease free water to 1 µl of the 100 µM 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 µl of nuclease free water. Make sure the new tube is labeled accordingly.</li>
	<li>Add 2 µl of Commercial Master Mix Buffer to the microcentrifuge tube.</li>
	<li>Add 1 µl of pGR-Blue plasmid (35-50 ng/µl) to the microcentrifuge tube.</li>
	<li>Add 2 µl of the 40 nM annealed terminators to the microcentrifuge tube.</li>
	<li>Add 1 µl 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 °C followed by 1 cycle of 15 min at 55 °C. Use samples immediately or freeze and store at -20 °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/µg ) chemically competent E. coli (JM109) cells for transformation.9 For best results, add 3-5 µl of the ligation reaction to 50 µl of competent cells. When using chemical competent cells, follow the transformation protocol specific to the cells being used.</li>
	<li>Spread half (25 µl) of the transformed cells onto pre-warmed LB (Amp/arb) agar plates using a sterile L-shaped "hockey stick" and incubate O/N at 37 °C. Due to ampicillin's rapid rate of decomposition, do not incubate at 37 °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 °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 µl 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 µl 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 °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="Fluorescence/optical density (OD600) formula; microbiology data analysis; experimental equation." src="/files/ftp_upload/54064/54064eq1.jpg" data-altupdatedat="1788235205000" data-alt="Equation 1"> for both GFP and RFP.</li>
	<li>After normalization, determine relative terminator strength using the equation  <img alt="Gene expression ratio formula; GFP and RFP synthesis balance; equation in molecular biology research." src="/files/ftp_upload/54064/54064eq2.jpg" data-altupdatedat="1788235205000" data-alt="Equation 2"></li>
</ol>

<ol><li>Li, J., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relationship+between+promoter+sequence+and+its+strength+in+gene+expression%5BTitle%5D)+AND+%22Eur+Phys+J+E+Soft+Matter%22%5BJournal%5D)">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/pubmed?term=((A+one+pot%2C+one+step%2C+precision+cloning+method+with+high+throughput+capability%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((GreenGate---a+novel%2C+versatile%2C+and+efficient+cloning+system+for+plant+transgenesis%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+582+natural+and+synthetic+terminators+and+quantification+of+their+design+constraints%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">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/pubmed?term=((Vision+and+change+in+biology+undergraduate+education%2C+a+call+for+action--initial+responses%5BTitle%5D)+AND+%22CBE+Life+Sci+Educ%22%5BJournal%5D)">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/pubmed?term=((Vision+and+change+in+the+biology+community%3A+snapshots+of+change%5BTitle%5D)+AND+%22CBE+Life+Sci+Educ%22%5BJournal%5D)">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/pubmed?term=((ARNold%3A+a+web+tool+for+the+prediction+of+Rho-independent+transcription+terminators%5BTitle%5D)+AND+%22RNA+Biol%22%5BJournal%5D)">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/pubmed?term=((pClone%3A+Synthetic+Biology+Tool+Makes+Promoter+Research+Accessible+to+Beginning+Biology+Students%5BTitle%5D)+AND+%22CBE+Life+Sci+Educ%22%5BJournal%5D)">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/pubmed?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%3A%3ASPA%5BTitle%5D)+AND+%22J+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Constitutive+mutations+in+the+Escherichia+coli+AraC+protein%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">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/pubmed?term=((Regulation+of+the+L-arabinose+operon+of+Escherichia+coli%5BTitle%5D)+AND+%22Trends+Genet%22%5BJournal%5D)">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/pubmed?term=((Composability+of+regulatory+sequences+controlling+transcription+and+translation+in+Escherichia+coli%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">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/pubmed?term=((Inferring+gene+regulatory+logic+from+high-throughput+measurements+of+thousands+of+systematically+designed+promoters%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">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 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><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

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 °C (Figure 3).
After colony selection, a plate...

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 ...

The overall goal of the synthetic biology protocol is to rapidly determine terminator functionality and quantify terminator strength in an in-vitro system that utilizes golden gate assembly. This protocol is used to test terminators that we found in silico using bio-informatics and test them in a biological setting. This allows for rapid and simple confirmation of bio-informatically identified terminators in a lab bench setting.The main advantage of this protocol is its speed. We can go from bio-informatic identification to biological confirmation in less than one week. And in doing so, we can also verify that a terminator is in fact a terminator and we can quantify its terminating strength.Generally, individuals will initially struggle with this technique, because it sounds counterintuitive to add the restriction enzyme with a ligase in the same tube. However we use type 2S restriction endonucleases and the recognition site for the enzyme is separate from the actual cut site. Once the terminator is ligated into the pGR blue plasmid, this is a permanent ligation.However, by having the restricton enzyme and the ligase in the same tube, going through multiple cycles, we can achieve a high cloing efficiency. Begin this procedure by re-suspending the individual oligonucleotides in nuclease-free water at a concentration of 100 micromolar, then prepare 10x annealing buffer. Next, label a 1.5 millilter micro-centrifuge tube for each terminator to be tested.To each tube, add 16 microliters of ultra-pure water, 2 microliters of 10x annealing buffer, and 1 micro liter each of the top and bottom strand oligonuclotides from the desired terminators. It's very important to make sure that the oligonuclotides you ordered have the correct sticky ends. Because once they're annealed together, it is important to ensure that the terminator is in the correct orientation.If the terminator's in the wrong orientation, pGR blue will not be able to verify if it's a functioning terminator. Next place the micro-centrifuge tubes in a float and boil them for four minutes in a water bath. After four minutes, turn off the heat plate but leave the tube in the water overnight to slowly cool.Store the annealed terminators at 20 degrees Celsius. To make a 40 nanomolar dilution of the annealed terminators, first thaw them on ice, then in a new 1.5-milliliter micro-centrifuge tube, add 124 microliters of nuclease-free water, to 1 microliter of the annealed terminators. Centrifuge the samples for 30 seconds at 10, 000x g.After the spin, store the samples on ice. In a new thermocycler appropriate tube, add 6 microliters of nuclease-free water. Then add one microliter of commercial DNA ligase buffer.Perform all of the subsequent steps on ice. Next, add 1 microliter containing 35 to 50 nanograms per microliter of the pGR blue destination plasmid, 1 microliter of the 40 nanomolar annealed terminators, 0.5 microliters of commercial high-fidelity BSA1 restriction endonuclease, and 0.5 microliters of commercial DNA ligase. Centrifuge the reaction mixture at 10, 000x g for 120 seconds.Then place the tube directly in the thermocycler and run the following program. 20 cycles of 1 minute at 37 degrees Celsius, followed by 1 minute at 16 degrees Celsius and then one cycle of 15 minutes at 37 degrees Celsius. Following the run, either freeze and store the samples at 20 degrees Celsius, or proceed immediately to transformation.Transform the ligation reactions into chemically competent e. coli cells using any appropriate transformation protocol. Here, JM109 cells are transformed using zippy transformation.Using an L-shaped glass hockey stick, spread 25 microliters of the transformed cells onto pre-warmed LB agar plates containing 1 millimolar ampicillin, and 10 millimolar arabinose. Incubate the plates overnight at 37 degrees Celcius. Do not incubate them for more than 24 hours.Ampicillin degrades rapidly. The next day, inspect the color of the colonies. A successful ligation should be white or yellow in visible light, and will fluoresce green under blue or UV light.An unsuccessful ligation will produce a colony that is blue in color under visible light. For each of the terminators and controls, prepare a test tube containing 5 milliliters of sterile LB broth with 1 millimolar ampicillin and 10 millimolar arabinose. When performing this protocol, with pGR it's really important to ensure that the proper controls are in place.The first control should just be LB broth. The next control should be cells that have resistance to ampicillin, but do not fluoresce under blue light. These will serve as a blank.The next control is a pGR plasmid that is not cut so it has no terminator in place and this will serve as the baseline for terminator strength and the last one of your tubes will be for your sample. Under visible light, use a sterile loop to pick 2 to 3 white or yellow colonies, and transfer them to the LB broth. Allow the samples to incubate on a shaker at 37 degrees Celsius at 160 rpm for 16 to 18 hours.The next day, use a spectrophotometer to determine the optical cell density of each sample at 600 nanometers. Generally, after this amount of time, an OD 600 of 0.8 to 1.0 is expected. Place a sterile 96 well plate into the hood.For each sample or control, prepare 3 wells with 199 microliters of sterile LB broth with ampicillin and arabinose. Next, pipette 1 microliter of the cell solution from the overnight broths into individual wells of the 96 well plate. Fill the plates with a breathable cover and then shake them for 18 to 20 hours at 37 degrees Celsius and 160 rpm.The next day, use a plate reader to determine the OD 600, which should be approximately 0.5. Also, measure the G of P fluorescence at an excitation wavelength of 395 nanometers and at emission of 509 nanometers. Measure RFP fluorescence at an excitation wavelength of 575 nanometers and emission of 605 nanometers.Using Excel, normalize for the variation in growth by dividing the florescence by the OD 600. After normalization, determine the relative terminator strength by taking the GFP terminator fluorescence over GFP control fluorescence and dividing it by the RFP terminator fluorescence over the RFP control fluorescence. Fix putative terminators T1 through 6 bio-informatically identified in the microbacteria phage bernal 13 were analyzed as described in this video.These 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. To determine the best reference control for quantifying terminator strength, raw data using the original pGR plasmid containing no terminator, and pGR blue cloned with a sequence known not to be a terminator, were compared.As can be seen here, both controls showed similar levels of GFP and RFP expression and more importantly, produced similar GFP to RFP ratios. All of these results represent individual colonies that were grown in triplicate and averaged for visual representation. Once mastered, this technique can take as little as 72 hours including shipping and ordering time.It is important to note that pGR blue only works in e.coli. Additionally, since we're only looking at a small piece of the terminator, we really have no idea of other regulatory elements that may control transcription. To verify the results you achieve with this protocol using pGR blue, additional methods can be used like quantitative PCR.Additionally, the pGR insert can be cut out and ligated into a plasmid of your choice.

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Research

JoVE Journal

Biology

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.

Mouse Genome Engineering Using Designer Nucleases

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.

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

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.

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

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 β-lactamase selected at a clinically relevant dosage of ampicillin are provided.

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

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.

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

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.

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

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

We describe a method to generate saturating transposon mutant libraries in Gram-negative bacteria and subsequent preparation of DNA amplicon libraries for high-throughput sequencing. As an example, we focus on the ESKAPE pathogen, Acinetobacter baumannii, but this protocol is amenable to a wide range of Gram-negative organisms.

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

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.

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

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.

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

Discover rapid terminator verification using Golden Gate Assembly with the pGR-Blue plasmid. This synthetic biology method quantifies terminator strength in vitro within one week.

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

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