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| <li id="b1">Annealing Oligos</li> | | <li id="b1">Annealing Oligos</li> |
| <li id="b2">BP Reaction</li> | | <li id="b2">BP Reaction</li> |
- | <li id="b3"> Capped Transcription Reaction Assembly</li>
| |
| <li id="b4"> Cell Stock </li> | | <li id="b4"> Cell Stock </li> |
| <li id="b5"> Gel Extraction - QIAGEN</li> | | <li id="b5"> Gel Extraction - QIAGEN</li> |
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| </ul> | | </ul> |
| </div><!-- end bacterial --> | | </div><!-- end bacterial --> |
- |
| |
- | <h3><a href="#">In Vitro</a></h3>
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- |
| |
- | <div class="col_list">
| |
- | <ul>
| |
- | <li id="iv1">Gate Anneals</li>
| |
- | <li id="iv2">Plate Reader Studies</li>
| |
- | <li id="iv3">In Vitro Transcription - New England BioLabs </li>
| |
- |
| |
- | </ul>
| |
- | </div><!-- end invitro-->
| |
| | | |
| <h3><a href="#">Mammalian</a></h3> | | <h3><a href="#">Mammalian</a></h3> |
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| </ul> | | </ul> |
| </div><!-- instrumentation--> | | </div><!-- instrumentation--> |
- |
| |
- | <h3><a href="#">Automation</a></h3>
| |
- |
| |
- | <div class="col_list">
| |
- | <ul>
| |
- | <li id="robot"> Automation</li>
| |
- | </ul>
| |
- | </div><!-- instrumentation-->
| |
- |
| |
- |
| |
- | </div>
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| | | |
| </div><!--end col_nav--> | | </div><!--end col_nav--> |
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| <div class="section" id="headerbio"> | | <div class="section" id="headerbio"> |
| <h1> Methods and Protocols Overview</h1> | | <h1> Methods and Protocols Overview</h1> |
- | In order to assess the functionality of RNA strand displacement <em>in vivo</em>, as well as to design and implement our sensing, processing, and actuating modules for our proposed genetic circuit, our team employed a variety of protocols and methods in our experimentation. | + | In order to assess the functionality of exosome-mediated cell-cell communication <em>in vivo</em>, as well as to design and implement our various modules for our proposed genetic circuits, our team employed a variety of protocols and methods in our experimentation. |
- | The experiments that we conducted to produce our results included the generation of nucleic-acid circuitry components in <em>E. coli</em> bacteria, the <em>in vitro</em> construction and testing of strand displacement components, the <em>in vivo</em> testing of various circuit components in HEK293 mammalian cells, and the use of laboratory instrumentation for data generation and analysis. | + | The experiments that we conducted to produce our results included the generation of nucleic-acid circuitry components in <em>E. coli</em> bacteria, the <em>in vitro</em> construction and testing of components, the <em>in vivo</em> testing of various circuit components in HEK293 and Jurkat mammalian cells, and the use of laboratory instrumentation for data generation and analysis. |
| <br/><br/> | | <br/><br/> |
| <img src='https://static.igem.org/mediawiki/2012/8/8e/WilsonMIT.jpg' style="width: 180px"> | | <img src='https://static.igem.org/mediawiki/2012/8/8e/WilsonMIT.jpg' style="width: 180px"> |
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| <LI> Add 1 uL of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37°C for 10 minutes. </LI> | | <LI> Add 1 uL of the Proteinase K solution to each sample to terminate the reaction. Vortex briefly. Incubate samples at 37°C for 10 minutes. </LI> |
| <LI> Transform 1 uL of each BP reaction into competent cells (see transformation protocol). Plate 20 uL and 100 uL of each on LB plates containing 100 ug/mL kanamycin. </LI> | | <LI> Transform 1 uL of each BP reaction into competent cells (see transformation protocol). Plate 20 uL and 100 uL of each on LB plates containing 100 ug/mL kanamycin. </LI> |
- | </OL>
| |
- | <div class="clear"></div>
| |
- | </div>
| |
- |
| |
- | <div class="section" id="b3bio">
| |
- | <h1> Capped Transcription Reaction Assembly </h1>
| |
- | <OL>
| |
- | <LI> Use mMESSAGE mMACHINE Kit: thaw the frozen reagents [on ice]. Place the RNA Polymerase Enzyme Mix on ice, it is stored in glycerol and will not be frozen at -20°C. Vortex the 10X Reaction Buffer and the 2X NTP/CAP until they are completely in solution. Once thawed, store the ribonucleotides (2X NTP/CAP) on ice, but keep the 10X Reaction Buffer at room temperature while assembling the reaction. All reagents should be microfuged briefly before opening to prevent loss and/or contamination of material that may be present around the rim of the tube. </LI>
| |
- | <LI>Assemble transcription reaction at room temp. The spermidine in the 10X Reaction Buffer can coprecipitate the template DNA if the reaction is assembled on ice. Add the 10X Reaction Buffer after the water and the ribonucleotides are already in the tube. Add the 10X Reaction Buffer after the water and the ribonucleotides are already in the tube. The following amounts are for a single 20 uL reaction. Reactions may be scaled up or down if desired. </LI>
| |
- | <LI> IMPORTANT: the following reaction setup is recommended when the RNA produced will be 300 bp - 5 kb in length. For transcripts longer or shorter than this, see optimization protocols.
| |
- |
| |
- | <LI> To 20 uL Nuclease-free Water add: L 2x NTP/CAP, 2 uL 10X Reaction Buffer, 0.1-1 ug Linear template DNA (use 0.1-0.2 ug PCR-product template or 1 ug linearized plasmid template), 2 uL Enzyme Mix. </LI>
| |
- |
| |
- | <LI> Mix thoroughly. Gently flick the tube or pipette the mixture up and down gently, and then microfuge tube briefly to collect the reaction mixture at the bottom of the tube. </LI>
| |
- |
| |
- | <LI>Incubate at 37C, 1 hr. Typically 80% yield is achieved after a 1 hr incubation. For maximum yield, we recommend a 2 hr incubation. Since SP6 reactions are somewhat slower than T3 and T7 reactions, they especially may benefit from the second hour of incubation. A second hour of incubation is recommended for synthesis of <300 base transcripts and for inefficiently transcribed templates. If the reaction is trace-labelled: after the incubation (before or after TURBO DNase treatment), remove an aliquot of trace-radiolabeled reactions to assess yield by TCA precipitation. </LI>
| |
- |
| |
- | <LI> (Optional) Add 1 uL TURBO DNase, mix well and incubate 15 min at 37 C. This DNase treatment removes the template DNA. For many applications it may not be necessary because the template DNA will be present at a very low concentration relative to the RNA. </LI>
| |
| </OL> | | </OL> |
| <div class="clear"></div> | | <div class="clear"></div> |
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| <LI> Place plate upside down overnight and let incubate at 37°C (~ 16 hrs). Should see colonies if successful. </LI> | | <LI> Place plate upside down overnight and let incubate at 37°C (~ 16 hrs). Should see colonies if successful. </LI> |
| </OL> | | </OL> |
- | <div class="clear"></div>
| |
- | </div>
| |
- |
| |
- |
| |
- | <div class= "section" id="iv1bio">
| |
- | <h1> Gate Anneals </h1>
| |
- | <em>Annealing oligonucleotides for strand displacement</em><br/><br/>
| |
- | One reaction results in 100 µL of annealed product at 10 µM (calculated based on the bottom strand) in 1X TAE buffer (12.5 mM Mg<sup>2+</sup>).<br/>
| |
- | <br/>
| |
- | <em>Note</em>: The top strand refers to an output strand that can act as a downstream input, whereas the bottom strand refers to the gate strand, which is always complexed with another strand in reactions.<br/>
| |
- | <br/>
| |
- | <ol>
| |
- | <li>In a PCR tube mix:
| |
- | <ul>
| |
- | <li>12 µL of top strand (from a 100 µM stock)</li>
| |
- | <li>10 µL of bottom strand (from a 100 µM stock)</li>
| |
- | <li>5 µL of 10x TAE buffer (125 mM Mg<sup>2+</sup>)</li>
| |
- | <li>23 µL of nuclease free water</li>
| |
- | </ul>
| |
- | </li>
| |
- | <li>Run a PCR program that starts at holds at 92°C for 2 minutes and then decreases the temperature by 0.1°C over (every) 6 seconds until reaching 22°C.</li>
| |
- | <li>Add 50 µL of 1X TAE buffer (12.5 mM Mg<sup>2+</sup>).</li>
| |
- | <li>Store at -20°C.</li>
| |
- | </ol>
| |
- | <br/>
| |
- | <em>Note</em>: If the products are fluorescent, use an opaque tube or cover the tube in foil in order to protect it from being bleached by light.<br/>
| |
- | <br/>
| |
- | Adapted from Qian L, Winfree E. <em>Scaling up digital circuit computation with DNA displacement cascades.</em>, Science. 2011 Jun 3; 332 (6034):1196-201.<br/>
| |
- |
| |
- | <div class="clear"></div>
| |
- | </div>
| |
- |
| |
- | <div class= "section" id="iv2bio">
| |
- | <h1> Plate Reader Studies </h1>
| |
- | <h2><b> Kinetic Studies</b> </h2>
| |
- |
| |
- | <p>
| |
- | <em>Note</em>: Buffer is 1X TAE buffer (12.5 mM Mg<sup>2+</sup>). Carrier is TTT-TTT-TTT-TTT-TTT-TTT-TT (DNA), in buffer. Amount of reporter is determined by the amount of fluorescent strand. 1X reactive species concentration = 10 nM = 1 pmol in 100 µL.
| |
- | </p>
| |
- |
| |
- | <ol>
| |
- | <li>In a 96-well plate add 500 pmol carrier to each well being used.
| |
- | Suggested: 50 µL of 10 µM stock (in 1X buffer).
| |
- | <li>Let wells incubate with carrier for a few minutes.
| |
- | Suggested: 10 minutes.
| |
- | <li>Prepare the following wells by adding:
| |
- | <ul>
| |
- | <li>Blank: 50 µL of buffer.</li>
| |
- | <li>Positive control: 1 pmol RNA-ROX (reporter bottom strand) or dsROX (reporter bottom strand annealed to an input strand).<br/>
| |
- | <em>Suggested</em>: 1 µL of 1 µM stock, 49 µL buffer.</li>
| |
- | <li>Baseline (optional): 1.2 pmol reporter complex.<br/>
| |
- | <em>Suggested</em>: 1.2 µL of 1 µM stock, 48.8 µL buffer.</li>
| |
- | </ul>
| |
- | <br/>
| |
- | Blank, positive control and baseline wells should be at 100 µL volume.<br/>
| |
- | <br/>
| |
- | <ul>
| |
- | <li>Negative control: 1.2 pmol reporter complex.</li>
| |
- | <li>Reaction well: 1.2 pmol reporter complex.<br/>
| |
- | <em>Suggested</em>: add 1.2 µL of 1 µM stock, 47.8 µL buffer.<br/>
| |
- | Negative and reaction wells should be at 99 µL volume.</li>
| |
- | </ul>
| |
- | <li>Measure the baseline fluorescence (10-20 datapoints as desired).</li>
| |
- | <li>Add inputs.<br/>
| |
- | <em>Suggested</em>: 1X = 1 µL at 1 µM.</li>
| |
- | <li>Mix with “magical” pipette tip:
| |
- | <ol>
| |
- | <li>Rinse pipette in 10 µM carrier solution by pipetting up and down.</li>
| |
- | <li>Use this empty tip to pipette up and down in the well.</li>
| |
- | <li>Use a new tip for each well.</li>
| |
- | </ol>
| |
- | <li>Measure fluorescence<br/>
| |
- | <em>Suggested</em>: For experiments lasting hours, 3 minutes between each sampling is sufficient. For larger concentrations decrease this time to see faster kinetics. Avoid oversampling as this can cause photobleaching.</li>
| |
- | </ol>
| |
- |
| |
- | <p>
| |
- | <em>Note</em>: If there are more components than just input and reporter, add necessary controls (+/- input). Adjust volumes of buffer accordingly.
| |
- | </p>
| |
- | <p>
| |
- | The reporter is added in excess (1.2x) to guarantee that the reporting reaction is driven to maximum completion, resulting in 1x fluorescent output from 1x input.</p>
| |
- | <p>
| |
- | Carrier is added to prevent non-specific sticking of RNA or DNA to plastic, such as pipette tips or plate wells (Zhang, D., Turberfield, A., Yurke, B., Winfree, E. <em>Engineering Entropy-Driven Reactions and Networks Catalyzed by DNA.</em>, Science 2007 Nov; 318 (5853):1121-1125).
| |
- | </p>
| |
- | <p>
| |
- | Data was gathered using the Tecan Safire II Microplate Reader with Magellan 5.03 software.<br/>
| |
- | Settings for ROX fluorophore: excitation 588 nm, emission 611 nm, bandwith (ex/em) 10 nm, gain 160, 5 flashes per read, 40 µs integration time, 0 µs lag time, Z-position 6286 nm (top, 96 well plate), 100 ms between move and flash.<br/>
| |
- | Settings for Alexa fluorophore: excitation 495 nm, emission 517 nm, bandwith (ex/em) 10 nm, gain 120, 5 flashes per read, 40 µs integration time, 0 µs lag time, Z-position 6286 nm (top, 96 well plate), 100 ms between move and flash.<br/>
| |
- | Data analysis was performed using standard spreadsheet software.
| |
- | </p>
| |
- |
| |
- | <div class="clear"></div>
| |
- | </div>
| |
- |
| |
- | <div class= "section" id="iv3bio">
| |
- | <h1> In Vitro Transcription - New England BioLabs </h1>
| |
- | <OL>
| |
- | <LI> Thaw the 10X transcription buffer and 20X NTP mix at room temperature for the minimum amount of time required for complete thawing. Do not thaw at 37°C. If precipitant is evident following thawing, vortex briefly to resuspend. Keep the 20X High Molecular Weight Component (HMW) Mix and T7 RNA Polymerase at –20°C until needed. </LI>
| |
- | <LI> Combine the following, in order, taking caution to avoid ribonuclease contamination. Set up separate reactions for each plasmid of interest: RNase-Free Water to 40 uL, 4 uL10X Transcription Buffer 4, 2uL 20X Ribonucleotide Solution Mix, 2uL Template(s) (1–2 µg) X µl, 20X HMW Mix, 2 uL T7 RNA Polymerase (500 units/µl) </LI>
| |
- | <LI> Incubate at 37 °C for 2-4hrs. </LI>
| |
- | <LI> For analysis of IVT run 1uL of transcription reaction in a 1% agarose gel. </LI>
| |
- |
| |
- | Adapted from New Englands Biolabs protocol, which can be found at http://www.neb.com/nebecomm/ManualFiles/manualE2030.pdf.
| |
- | </OL>
| |
- |
| |
| <div class="clear"></div> | | <div class="clear"></div> |
| </div> | | </div> |
| | | |
| <div class= "section" id="m1bio"> | | <div class= "section" id="m1bio"> |
- | <h1> DNA Transfection </h1> | + | <h1> DNA Transfection of HEK293 - Lipofectamine 2000</h1> |
| <OL> | | <OL> |
| <LI>Label enough eppendorf tubes for the number of conditions/wells you are transfecting plus one for a master mix of transfection reagent and unsupplemented DMEM. If possible, try to pool conditions/wells, i.e, if doing a small molecule induction ladder.</LI> | | <LI>Label enough eppendorf tubes for the number of conditions/wells you are transfecting plus one for a master mix of transfection reagent and unsupplemented DMEM. If possible, try to pool conditions/wells, i.e, if doing a small molecule induction ladder.</LI> |
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| All flow cytometry observations were carried out using a LSRFortessa flow analyzers (BD Biosciences) with the following sets of setting. Alexa and GFP/YFP were measured using a 488 nm Laser, a 505 nm Longpass filter and a 530/30 emission filter in the linear range of PMT values adjusted such that negative cells fell at 10<sup>2</sup>. BFP was measured with a 405 nm Laser, a 460 nm Longpass filter and a 480/40 emission filter. mKate and ROX were measured using a 561 nm laser and a 582/15 emission filter. For each sample, cells were trypsinized according to details in cell culture protocols and approximately 1 x 10<sup>4</sup> to ~ 1 x 10<sup>5</sup> cell events were collected. In parallel, Rainbow Calibration Particles (BD Biosciences) were measured in order to equalize the data between different instruments and settings. | | All flow cytometry observations were carried out using a LSRFortessa flow analyzers (BD Biosciences) with the following sets of setting. Alexa and GFP/YFP were measured using a 488 nm Laser, a 505 nm Longpass filter and a 530/30 emission filter in the linear range of PMT values adjusted such that negative cells fell at 10<sup>2</sup>. BFP was measured with a 405 nm Laser, a 460 nm Longpass filter and a 480/40 emission filter. mKate and ROX were measured using a 561 nm laser and a 582/15 emission filter. For each sample, cells were trypsinized according to details in cell culture protocols and approximately 1 x 10<sup>4</sup> to ~ 1 x 10<sup>5</sup> cell events were collected. In parallel, Rainbow Calibration Particles (BD Biosciences) were measured in order to equalize the data between different instruments and settings. |
| </p> | | </p> |
- | </div>
| |
- |
| |
- | <div class="section" id="robotbio">
| |
- | <h1>Robotic Automation</h1>
| |
- | <p>
| |
- | Members of our team worked with a TECAN EVO 150 liquid handling robot to automate common biology tasks for iGEM and for others in our labspace. We worked on a set of Python modules that offer more advanced control of the robot than the default TECAN programming language. Along the way, we automated the Miniprep DNA separation protocol, set up automated transfections, and wrote a program to compile spreadsheet input into robot code. The video below shows the robot running one of our protocols.
| |
- | </p>
| |
- | <p>
| |
- | <iframe width="420" height="315" src="http://www.youtube.com/embed/ymCVgEk6nI0" frameborder="0" allowfullscreen></iframe>
| |
- | <br />
| |
- | <i>Robot at work. Video is playing at 8x speed.</i>
| |
- | </p>
| |
- |
| |
| </div> | | </div> |
| | | |