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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<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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<p>      8. Results can now be analyzed. </p>
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<h3>PCR (Polymerase Chain Reaction) for Parts:</h3>
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<p>Ingredients: </p>
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<p>- 2.5μL Part Forward Primer</p>
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<p>- 2.5μL Part Reverse Primer</p>
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<p>- 0.1μL G-block / DNA template</p>
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<p>- 25μL Q5 2x High-Fidelity MasterMix</p>
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<p>- 19.9μL NFW (nuclease free water)</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 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>        Step 1: 98°C for 30 sec</p>
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<p>         Step 2: 98°C for 10 sec</p>
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<p>        Step 3: 70°C for 20 sec</p>
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<p>        Step 4: 72°C for 20-30sec/kilobase (typically)</p>
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<p>        Step 5: Enter “Go To” and then Step 2 and repeat for 25 cycles (or “times”)</p>
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<p>        Step 6: 72°C for 2 min</p>
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<p>        Step 7: 4°C for ∞ (Set to 00:00:00)</p>
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<p>        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>
+
−
<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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<h3>Introduction </h3>
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<p>Ingredients/Materials: *</p>
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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>
+
<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>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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<h3>PCR Check for Golden Gate Plasmids (multiple can be done at a time in different tubes)</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>- 0.5μL V part forward sequencing primer</p>
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<h4>Methods </h4>
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<p>- 0.5μL V part reverse sequencing primer</p>
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<br>
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<p>- 0.1μL GG Assembly</p>
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<h3>Testing spacers <i>in vivo</i></h3>
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<p>- 5μL 2x 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>- 3.9μL 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. Mix above ingredients in mini microfuge tube(s). </p>
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<p>      2. Spin in centrifuge.</p>
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<p>      3. Put it in thermocycler, process it in thermocycler as follows: </p>
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<p>            Step 1: 98°C for 30 sec</p>
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<p>            Step 2: 98°C for 10 sec</p>
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<p>            Step 3: 56°C for 20 sec</p>
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<p>            Step 4: 72°C for 20-30sec/kilobase (typically)</p>
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<p>            Step 5: Enter “Go To” and then Step 2 and repeat for 25 cycles (or “times”)</p>
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<p>            Step 6: 72°C for 2 min</p>
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<p>            Step 7: 4°C for ∞ (Set to 00:00:00)</p>
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<p>            Step 8: End</p>
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<h3>Making LB Media (& Autoclaving)</p>
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<h3> Testing dCas9 clamps <i>in vitro</i> </h3>
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<p>Ingredients:</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>-LB powder
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<p>-Distilled water
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<p>Materials:</p>
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<p>-Scale</p>
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<p>-Glass bottle</p>
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<p>-Autoclave</p>
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<p>Directions:</p>
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<p>      1. Measure out 5g LB powder. </p>
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<p>      2. Add LB powder to the bottle. </p>
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<p>      3. Add 250mL distilled water to bottle. </p>
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<p>      4. Autoclave for ~30 min. </p>
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<p>      To Autoclave:
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<p>              a. Fill autoclave with water until it covers metal ring. </p>
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<p>              b. Place metal bucket in autoclave. Put in metal dish. Place bottle (or item(s) to autoclave) at center. </p>
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<p>              c. Put lid on with the dangly metal cord in place. </p>
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<p>              d. Bolt on bolts tightly to seal the lid. </p>
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<p>              e. Open both manual release valves. </p>
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<p>             f. Plug in and turn on switch. </p>
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<p>              g. Wait until steam comes out of valve and wait 5 more minutes. </p>
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<p>              h. Close both valves and wait for temperature to rise. </p>
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<p>              i. Switch off the autoclave before it reaches red at 0.15. </p>
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<p>              j. Maintain between 0.1 and 0.5 for 30 min. </p>
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<p>              k. After complete, switch off and wait until pressure goes down back to 0. </p>
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<p>              l. Open manual valves and release steam. </p>
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<h3>Making LB Agar Plates w/ Antibiotic Resistance</p>
 
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<p>Ingredients:</p>
 
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<p>- 300ml H2O</p>
 
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<p>- 6g LB Powder</p>
 
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<p>- 4.5g Agar Powder</p>
 
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<p>- Antibiotic (usually use Kanamycin - 1μL per 1mL H2O)</p>
 
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<p>Materials: *</p>
 
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<p>- Autoclave</p>
 
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<p>- Scale</p>
 
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<p>- Glass bottle</p>
 
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<p>Directions: </p>
 
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<p>      1. Measure out the LB and Agar. Pour into a glass bottle and then pour in distilled water up to 300mL line. </p>
 
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<p>      2. Mix the above ingredients. </p>
 
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<p>      3. Autoclave for 30 minutes. </p>
 
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<p>      4. Cool down to around 50°C (~122°F). </p>
 
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<p>      5. Add 300μL Kanamycin (or chosen antibiotic, added accordingly).</p>
 
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<p>      6. Open lid to each plate carefully and pour plate near flame. </p>
 
  
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<h3>Bacterial Transformation of Plasmids (& Growing Liquid Cultures) </p>
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<br>
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<p>Ingredients/Materials: *</p>
+
<h4><a href="https://2016.igem.org/Team:Alverno_CA/Protocols"> Click Here To Access More Protocols</a></h4>
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<p>- DNA GG Plasmid Mixture</p>
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<p>- Competent Cells</p>
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<p>- SOC Media (or LB Media if SOC is contaminated…) </p>
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<p>- 2ml Microtubes</p>
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<p>- Tube Rack</p>
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<p>- Ice</p>
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<p>- Timer</p>
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<p>- 42°C Water Bath (set early on!) </p>
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<p>- 37°C Incubator (set early on!) </p>
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<p>- Petri Plates with LB agar and antibiotic</p>
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<p>- Sterile Spreader or sterile glass beads</p>
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<p>Directions: </p>
+
−
<p>      1. Thaw competent cells on ice. (If delicate, take out after step 3, and do step 2 after step 3) </p>
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<p>      2. Pipette 20μL of competent cells into 2mL tube.</p>
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<p>      3. Pipette 1μL of control DNA into 2mL tube.</p>
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<p>      4. Pipette 1μL of resuspended DNA into 2mL tube.</p>
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<p>      5. Close 2ml tubes and incubate on ice for 30 minutes. </p>
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<p>      6. Heat shock tubes at 42°C for 1 minute.</p>
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<p>      7. Incubate on ice for 5 minutes. </p>
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<p>      8. Pipette 200μL SOC media (or LB media) to each transformation.</p>
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<p>      9.  Incubate at 37°C for 2 hours—in incubator. </p>
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<p>      10. Pipette each transformation on petri plates (labelled!).</p>
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<p>      11. Incubate transformations overnight (14-18 hours) at 37°C. </p>
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<p>  Next Day: </p>
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<p>      12. Pick single colonies. Transfer each single colony to a gridded plate (labelled), dip pipette tip into PCR reaction to do a colony PCR to verify part size, then place tip into liquid culture to grow up liquid cell cultures. </p>
+
−
<p>      13. To do Colony PCR, create mixture according to PCR Check for Golden Gate Plasmids Protocol without 0.1uL of GG Assembly (or DNA Plasmid). Put in the MasterMix as well.
+
−
</p>
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<p>      14. After picking colony with pipette tip dip into reaction mixture and then put PCR reaction into the thermocycler and continue PCR protocol. </p>
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<p>      15. Incubate gridded plate and the liquid cultures at 37°C overnight. Take out the next morning and store in refrigerator (4°C). </p>
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<h3>Plate Reading (for Fluorescence, Absorbance, Induction, etc.)
 
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<p>Ingredients/Materials: *
 
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<p>- Liquid cultures
 
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<p>- 96 well plate (A-H by 1-12)
 
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<p>- Plate Reader (we use VICTOR X3)
 
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<p>- LB Media
 
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<p>Directions:
 
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<p>      1. Pipette in 100uL per well for each liquid culture. Place plate into reader. Set protocol to absorbance and hit run (green arrow button).
 
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<p>      2. Measure Absorbance at 600nm for all samples in all standard measurement modes in plate reader.
 
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<p>      3. Import data into Excel Sheet after run is finished. Use Normalization sheet tab and copy over. Enter data accordingly.
 
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<p>      4. Record the data, specifically volume of preloading culture and preloading media from the table in the notebook.
 
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<p>      5. Dilute accordingly (media is LB media with antibiotic) in a new 96 well plate (if needed). Usually 500uL per well.
 
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<p>      6. Place plate back into reader and set to OD-RFP-GFP protocol and start run.
 
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<p>      7. Let it run overnight (120 runs total with about 30 second intervals) and check in the morning.
 
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<p>      8. Import data results into Excel spreadsheet. Upload to Google drive.
 
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<p>      9. To analyze data using Python program—file must be in csv format. (If you would like the code for analyzing this type of data, please contact us!)
 
  
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<p>For any questions about Protocols, email: alverno.igem@gmail.com</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)

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