Difference between revisions of "Team:Alverno CA/Experiment"

 
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<head>
 
<head>
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<title>Alverno iGEM 2016</title>
 
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<link rel="icon" href="https://pbs.twimg.com/profile_images/2199338457/alverno-logo_high_400x400.PNG">
 
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</head>
 
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<body>
 
<body>
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<h1><center>Alverno iGEM 2016</center></h1>
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<br>
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<center><img src="https://s-media-cache-ak0.pinimg.com/originals/86/08/18/860818cfc65e5ff04725bb0f0c05a8af.png" alt="Alverno iGEM Logo" style="width:300px;"></center>
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<h1><center>Experiment & Protocol</center></h1>
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<center><h2>Experiment</h2></center>
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<p>      *Note: Pipettes are needed. </p>
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<h3>Making the Agarose Gel:</h3>
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<div class="demo" id="container">
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<p>Ingredients/Materials: *</p>
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        <center>
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<p>- 0.6g agarose</p>
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            <ul>
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<p>- 50mL TBE 1x</p>
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                <li style="list-style: none"><br>
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<p>- 5μL SYBRsafe / 2.5μL EcoStain (light- and heat-sensitive)</p>
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                <br>
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<p>- 250mL conical flask</p>
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                </li>
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<p>- scale </p>
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<p>- Microwave</p>
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                <li><img src=
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<p>- Gel mold</p>
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                "https://static.igem.org/mediawiki/2016/5/52/T--Alverno_CA--Kiki_benchling.jpeg"
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<p>Directions: </p>
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                style="height:215px;width:215px;" title=
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<p>    1. Weigh 0.6g of agarose into flask.</p>
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                "Using Benchling to design primers"/>
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<p>    2. Add 50mL of TBE 1x Buffer</p>
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                </li>
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<p>     3. Microwave agarose solution until dissolved for 1 min (take out halfway to swirl)</p>
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                <li><img src=
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<p>         (If the liquid looks distorted, agarose solution need to be microwaved more)</p>
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                "https://static.igem.org/mediawiki/2016/1/1e/Cultures_Experiment_page.jpeg"
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<p>4. Cool slightly and add SYBRsafe or EcoStain</p>
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                style="height:215px;width:215px;" title=
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<p>5. Pour gel into mold and put the combs into their spots</p>
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                "Liquid cultures of RFP/GFP constructs; transformed <i>E. coli</i>">
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<p>6. Cool until solidified.</p>
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                </li>
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<p><h3>Gel Electrophoresis & Screening the gel</h3></p>
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                <li><img src=
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<p>Ingredients/Materials: * </p>
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                "https://static.igem.org/mediawiki/2016/c/ca/T--Alverno_CA--Victoria_Plate_Reader2.jpg"
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<p>- TBE 1x Buffer</p>
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                style="height:215px;width:215px;" title=
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<p>- agarose gel (w/ right number of wells)</p>
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                "Victoria using the plate reader">
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<p>- DNA reaction (either parts=PUCTV, or PCR check for GG plasmids)</p>
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                </li>
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<p>- purple loading dye</p>
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</center>
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<p>- Gel box</p>
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</ul>
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<p>Directions:</p>
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<p>       1. Place agarose gel into gel box with wells on negative side. </p>
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    </div>
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<p>       2. Fill up gel box up with TBE 1x Buffer up to line or at least above gel. </p>
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<br><br><br><br><br><br><br><br><br><br>
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<p>       3. Pipette 6ul of 2-log ladder on side lanes (or given wells).</p>
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<p>      4. Pipette 1ul of purple loading dye on parafilm. Then pipette 5ul of DNA reaction and mix with loading dye on parafilm. (*For PCR Check: Directly pipette 2ul of purple loading dye into 10ul of PCR Check Reaction in microfuge tube)</p>
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<p>      5. Set to 6ul and pipette 6ul of mixed reaction with loading dye into selected wells according to drawn diagram. See gel picture. </p>
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<h3>Introduction </h3>
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<p>       6. Set machine to 175V and for 20 minutes. Make sure bubbles are appearing!</p>
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<p>      7. After running place gel onto transilluminator (must be off) in glove box. Place box over it with hole and then place iPad above it and set to either video or time-lapse. Then turn on transilluminator within closed glove box while videotaping. </p>
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<h5> It has been reported in the synthetic biology literature that the relative orientations and positions of genes physically placed near each other in multigene circuits may affect their expression. Specifically, it has been postulated that the issue of supercoiling may be responsible for this interference. Considering this, our intent was to demonstrate these effects on a series of plasmids containing both a RFP coding device, and a GFP coding device on a pSB1C3 pSB1C3 or <a href="https://www.addgene.org/66067/">DVK_AE</a> (derived from pSB1K3). In between the two genes on each plasmid, we placed one of several insulators to separate the genes. These included a 500bp spacer, a 1000 bp spacer, and a dCas9 clamp; each was designed to isolate and eliminate interference. Constructed plasmids were inserted into E. coli (DH5α). Ultimately, the goal of the experiment was to accurately demonstrate interference between two genes (RFP/GFP) on the same plasmid and, in addition, find an insulating mechanism to restrain the effects of possible supercoiling. (For more details, see <a href="https://2016.igem.org/Team:Alverno_CA/Design">Design</a>)
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<p>      8. Results can now be analyzed. </p>
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<h3>PCR (Polymerase Chain Reaction) for Parts:</h3>  
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<br>After constructing these plasmids, we grew transformed <i>E. coli</i> as overnight liquid cultures. </h5>
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<p>Ingredients: </p>
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<br><br>
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<p>- 2.5μL Part Forward Primer</p>
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<h4>Methods </h4>
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<p>- 2.5μL Part Reverse Primer</p>
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<br>
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<p>- 0.1μL G-block / DNA template</p>
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<h3>Testing spacers <i>in vivo</i></h3>
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<p>- 25μL Q5 2x High-Fidelity MasterMix</p>
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<h5>Cultures of strains containing plasmids with a 500bp spacer or 1000bp spacer were diluted to the same OD. RFP expression (in AFU), GFP expression (in AFU), and OD were measured using a <a href="http://www.perkinelmer.com/product/victor-x5-for-fl-lum-uv-trf-fp-2030-0050">VICTOR-X3</a> plate reader. Inducers - specifically ATc and IPTG - were added to the strains containing plasmids with the 1000bp spacer. (See <a href="https://2016.igem.org/Team:Alverno_CA/Protocols">"Plate Reading"</a> for more information) </h5>
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<p>- 19.9μL NFW (nuclease free water)</p>
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<p>  Materials: *</p>
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<h3> Testing dCas9 clamps <i>in vitro</i> </h3>
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<p>- Centrifuge</p>
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<h5> Cultures of strains containing plasmids with a dCas9 clamp site spacer were mini-prepped to extract plasmids. We designed and constructed gRNA plasmids and obtained dCas9 expression plasmids (<a href="https://www.addgene.org/48657/">DS-SPCasN</a>). All plasmids were mini-prepped and expressed in TX-TL <a href="http://www.openwetware.org/wiki/Biomolecular_Breadboards">TX-TL</a>, an <i>in vitro</i> prototyping technique which mimics cell environments for transcription and translation. The plasmids were tested with and without inducers (IPTG for RFP, and ATc for GFP). GFP and RFP were measured with the plate reader. (See <a href="https://2016.igem.org/Team:Alverno_CA/Protocols">"Plate Reading"</a> for more information)</h5>
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<p>- Thermocycler</p>
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<p>- Mini microfuge PCR tube(s)</p>
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<p>   Directions: </p>
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<p>1. Mix above ingredients listed together in microfuge PCR tube. (Notice to add MasterMix last.)</p>
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<p>2. Spin in centrifuge.</p>
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<p>3. Put in thermocycler. Process it in thermocycler as follows: </p>
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<p>    a. Step 1: 98°C for 30 sec</p>
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<p>    b. Step 2: 98°C for 10 sec</p>
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<p>    c. Step 3: 70°C for 20 sec</p>
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<p>    d. Step 4: 72°C for 20-30sec/kilobase (typically)</p>
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<p>    e. Step 5: Enter “Go To” and then Step 2 and repeat for 25 cycles (or “times”)</p>
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<p>    f. Step 6: 72°C for 2 min</p>
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<p>    g.Step 7: 4°C for ∞ (Set to 00:00:00)</p>
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<p>    h. Step 8: End</p>
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<h3>PCR Purification of DNA: </h3>
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<p>Ingredients/Materials: *</p>
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<p>- PCR reaction(s) (DNA Part(s))</p>
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<p>- Molecular Biology Kit, which includes: </p>
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<p>      - Buffer B3 (with pre-added isopropyl alcohol)</p>
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<p>      - Wash Solution (with pre-added ethanol)</p>
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<p>      - Elution Buffer</p>
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<p>      - EZ-10 column(s)</p>
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<p>      - 1.5mL microfuge tube(s)</p>
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<p>Directions: (for each PCR reaction)</p>
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<p>    1. Transfer PCR reaction mixture (usually 50ul, ranges from 35-50ul) to a 1.5mL microfuge tube and add 5 volumes (5 x amount of PCR reaction mixture) of Buffer B3 (with pre-added isopropyl alcohol). </p>
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<p>     2. Transfer above mixture to EZ-10 column and leave at room temperature for 2 minutes. Centrifuge at 10,000rpm for 2 minutes. </p>
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<p>    3. Remove/empty flow-through in bottom tube. Add 750ul of Wash Solution (with pre-added ethanol) and centrifuge at 10,000rpm for 2 minutes. </p>
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<p>    4. Repeat washing procedure (from Step 3, “Add 750ul of…”). Remove/empty flow-through again. Spin at 10,000rpm for an additional minute. Throw away bottom clear tube with any remaining liquid.</p>
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<p>    5. Place top tube with white filter into clean 1.5mL microfuge tube. Check for ethanol using pipette tip. </p>
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<p>    6. Add 30-50uL (usually 40uL) of Elution Buffer to center of tube. Incubate at room temperature for 2 minutes. Centrifuge at 10,000rpm for 2 minutes to elute DNA. </p>
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−
<p>    7. Store at -20 degrees Celsius, or nanodrop for concentration and for dilutions (see Parts Dilutions Protocol). </p>
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<h3>Parts Dilutions: </h3>
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<br>
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<p>Ingredients/Materials: *</p>
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<h4><a href="https://2016.igem.org/Team:Alverno_CA/Protocols"> Click Here To Access More Protocols</a></h4>
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<p>- NFW</p>
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<p>- PCR Purified DNA Part Reaction (nanodropped with concentration)</p>
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<p>- 1.5mL microfuge tube </p>
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<p>Directions: </p>
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<p>      1. Identify number of bases and the concentration (in ng/uL, which is basically ug/mL). </p>
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<p>      2. Plug in numbers (bases and concentration) into Promega Biomath Calculator to convert from ug/mL (or ng/uL) to pmol/uL </p>
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<p>          (http://www.promega.com/a/apps/biomath/index.html?calc=ugmlpmolul).</p>
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<p>      3. Multiply resulting number by 1000 and that is the concentration in nM. </p>
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<p>      4. Plug into dilution equation: C1*V1=(30nM)(V2), where C1 is the concentration in nM, and V2 is equal to the amount wanted (typically 10uL-20uL). Then solve for V1. </p>
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<p>      5. Put in the amount of V1 of selected Part in 1.5mL microfuge tube. </p>
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<p>      6. Subtract V1 from V2. Put this amount of NFW into the tube. </p>
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<p>      7. Centrifuge.</p>
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<p>      8. Store at -20 degrees Celsius. </p>
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<h3>Golden Gate Assembly for Plasmids (Example with Golden Gate Assembly Protocol for GG37-52; multiple GG Assembly for Plasmids can be done at a time in different tubes as seen in example)</h3>
 
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<p>Ingredients: (per Golden Gate Assembly)</p>
 
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<p>- 1μL: P part (i.e. P1a, P2a, P3a, P4a)</p>
 
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<p>- 1μL: UC part (i.e. UC1a, UC2a, …, UC8a, etc.) </p>
 
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<p>- 1μL: T part (i.e. T1a, T2a, T3a, T4a)</p>
 
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<p>- 1μL: V part (i.e. V19d, V1a, V2a, etc.)</p>
 
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<p>- 1μL: GFP Mut Parts: P1ab, P2ab, T3ab, T4ab (Note: for all parts with GFP, matches according to Part, see combos image for example; some do not have GFP Mut parts and so add 1ul to NFW amount instead) </p>
 
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<p>- 1.5μL: T4 Ligase Buffer</p>
 
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<p>- 0.15μL: 100x BSA Standard</p>
 
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<p>- 1μL: BsaI</p>
 
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<p>- 2μL: T4 Ligase (2M cohesive units)</p>
 
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<p>- 5.35μL NFW</p>
 
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<p>Materials: *</p>
 
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<p>- Centrifuge</p>
 
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<p>- Thermocycler</p>
 
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<p>- Mini microfuge tube(s)</p>
 
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<p>Directions: </p>
 
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<p>      1. Write down combos of plasmid(s) (Insert picture. 08/29/16 protocol by Melody Wu)</p>
 
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<p>      2. Mix above ingredients in labelled mini microfuge tube(s).</p>
 
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<p>      3. Spin down in centrifuge.</p>
 
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<p>      4. Put in thermocycler, process it in thermocycler as follows:</p>
 
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<p>            - Step 1: 37°C for 3 min</p>
 
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<p>            - Step 2: 16°C for 4 min</p>
 
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<p>            - Step 3: Go to Step 1 and repeat for 25 cycles</p>
 
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<p>            - Step 4: 50°C for 5 min</p>
 
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<p>            - Step 5: 80°C for 5 min</p>
 
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<p>            - Step 6: 4°C for ∞ (Set to 00:00:00)</p>
 
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<p>            - Step 7: End</p>
 
  
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Latest revision as of 20:02, 2 November 2016


Experiment













Introduction

It has been reported in the synthetic biology literature that the relative orientations and positions of genes physically placed near each other in multigene circuits may affect their expression. Specifically, it has been postulated that the issue of supercoiling may be responsible for this interference. Considering this, our intent was to demonstrate these effects on a series of plasmids containing both a RFP coding device, and a GFP coding device on a pSB1C3 pSB1C3 or DVK_AE (derived from pSB1K3). In between the two genes on each plasmid, we placed one of several insulators to separate the genes. These included a 500bp spacer, a 1000 bp spacer, and a dCas9 clamp; each was designed to isolate and eliminate interference. Constructed plasmids were inserted into E. coli (DH5α). Ultimately, the goal of the experiment was to accurately demonstrate interference between two genes (RFP/GFP) on the same plasmid and, in addition, find an insulating mechanism to restrain the effects of possible supercoiling. (For more details, see Design)
After constructing these plasmids, we grew transformed E. coli as overnight liquid cultures.


Methods


Testing spacers in vivo

Cultures of strains containing plasmids with a 500bp spacer or 1000bp spacer were diluted to the same OD. RFP expression (in AFU), GFP expression (in AFU), and OD were measured using a VICTOR-X3 plate reader. Inducers - specifically ATc and IPTG - were added to the strains containing plasmids with the 1000bp spacer. (See "Plate Reading" for more information)

Testing dCas9 clamps in vitro

Cultures of strains containing plasmids with a dCas9 clamp site spacer were mini-prepped to extract plasmids. We designed and constructed gRNA plasmids and obtained dCas9 expression plasmids (DS-SPCasN). All plasmids were mini-prepped and expressed in TX-TL TX-TL, an in vitro prototyping technique which mimics cell environments for transcription and translation. The plasmids were tested with and without inducers (IPTG for RFP, and ATc for GFP). GFP and RFP were measured with the plate reader. (See "Plate Reading" for more information)

Click Here To Access More Protocols