Difference between revisions of "Team:Arizona State/Notebook"

 
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<h1><ins> Notebook: Chris Connot </ins></h1>
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<h2>AHL Degredation: Ethanol/ISO TraR</h2>
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<h3>Wednesday, 9/20</h3>
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<p>Notes: This is the second run of testing the degradation of AHLs via the treatment of 2-Propanol and ETOH. The first was with LasR and the next will be with 2620 (Lux) if the synthetic AHLs can be found. The first protocol is for preparing necessary parts for the plate reader which will have a protocol added below the Prep Protocol</p>
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<p>Notes: This is the second run of testing the degradation of AHLs via the treatment of 2-Propanol and ETOH. The first was with LasR and the next will be with 2620 (Lux) if the synthetic AHLs can be found. The first protocol is for preparing necessary parts for the plate reader which will have a protocol added below the Prep Protocol. </p>
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<h3>Prep Protocol: </h3>
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<ol type="1">
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<li>Grabbed 6 15mL culture tubes</li>
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<li>Labeled 2 for Neg Sender, 2 for TraR #2, and 2 for Neg Receiver</li>
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<li>Using a serological tube, 25mL of LB/AMP was pipetted out, and approximately 4 mL was pipetted into each culture tube (more than 4 mL will cause for decreased growth capacity). </li>
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<li>A seperate 250mL flask was used for additional supernatant of neg sender, and 50mL of LB/AMP was also pippetted into the flask. </li>
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<li>Using the bacteria plates for neg sender/receiver (9/14/17) and TraR #2 (8/16/17) transformation (s). The cells were scraped off from each plate using a different tip each time (single colony) and ejected into the corresponding culture tubes. </li>
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<li>The tubes were then properly closed and a foil was placed over the flask</li>
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<li>The tubes and flask were then placed into the 37C shaking incubator for overnight growth.</li>
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<li>After incubation, the OD600 is taken for the GFP+, -REC and the Sender</li>
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<li>Calculated volumes of GFP, -REC and the Sender to give a concentration of .70 at a volume of 500 ul</li>
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<li>The appropriate volume of supernatant is taken from the overnight cultures (after being spun down in the centrifuge at max speed for 1 minute) and placed in a total volume (including supernatant) of 500 ul of fresh LB/Amp</li>
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<li>Where calculated volumes utilize the equation C1V1=C2V2</li>
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<li>Sender is spun down till clear in 50 ml conical vial </li>
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<li>a filter syringe is used to transport the supernatant into a new 50 ml conical vial </li>
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<li>The receiver (TraR) is now spun down in a 50 ml conical vial (4 ml) and supernatant is discarded and the cells are resuspended in 20 mL of LB/AMP. </li>
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<li>After all cells have been spun down, resuspended and transferred into new media it is time to prepare the treatment tubes of AHL (treated with chosen alcohol) </li>
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<li>1.5 ml tubes are used to create serial dilutions of the alcohol for concentrations 10^-1 to 10^-12 (choose range based on need)</li>
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<li>Powdered synthetic AHL's are used to create the diluted 1.5ml concentrations </li>
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<li>To each 1.5 ml tube (10^-1 to 10^-12) the chosen alcohol (i.e ETOH or 2-propanol) is added and the tubes are closed to allow treatment to take place over 15 minutes </li>
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<li>The ETOH or 2-Propanol is added at an amount that maintains a total volume concentration of 70% ETOH or 2-propanol</li>
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<li>Once the 15 minutes is over the tubes are opened and placed in a well ventilated incubator in order to assist in the evaporation of the alcohol and ethyl-acetate that the AHL's were suspended in </li>
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<li>Once all components have evaporated the solid AHL's are resuspended in the original volume of ethyl-acetate that the dilutions were made in and placed on the vortex to ensure that the AHL's re-dissolve</li>
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<li>Then the overnight induction plate is setup with the following layout</li>
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<li>In the table below Well A4-A12 each have equal parts 150ul of the labeled product (i.e GFP+/-Sender supernatant) The wells were filled from 500ml stocks made for each different component. </li>
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<li>For the wells from B1-F6 there was the following stocks made 500ul Receiver, 490 -Sender and 10ul AHL) </li>
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<li>The wells themselves received 300 ul out of the stocks made from each concentration </li>
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<li>For wells B1-C3 the 'U' stands for Untreated (AHLs were not treated with the alcohol),
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Wells C4-D12 the 'I' stands for Isopropanol and cells D13-F-6 the 'E' stands for ethyl alcohol (EtOH). </li>
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<li>The well plate is then placed in the appropriate imaging machine and OD600 and GFP expression is measured over a 8-16 hr period depending on the length of data collection needed. </li>
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<center><img src=https://static.igem.org/mediawiki/2017/1/18/CC_graph.png></center>
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<h2>ESA Induction Plates Liquid/Plated</h2>
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<h3>Wednesday, 7/19</h3>
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<p>will be starting with 3 ul of solution for the liquid culture plates for each of the ESA/-REC/GFP+/2620</p>
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<p>finale amount used was 9 ul </p>
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<ul>
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<li>Liquid culture made @ 9:42 am </li>
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<li>ESA Plated Culture: Plated at 10:08 am </li>
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<li>-Sender Plate (was improvised) and plated at 10:30 am</li>
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<li>Liquid culture checked @ 10:51 am</li>
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<li>ESA Plated Culture: Checked at 11:08 am</li>
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<li>-Sender Plate (was improvised) and Checked at 11:32 am</li>
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<li>Liquid culture checked @ 11:51 am</li>
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<li>ESA Plated Culture: Checked at 12:08 pm</li>
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<li>-Sender Plate (was improvised) and Checked at 12:32 pm</li>
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<li>Liquid culture checked @ 12:58 pm</li>
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<li>ESA Plated Culture: Checked at 12:59 pm</li>
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<li>-Sender Plate (was improvised) and Checked at 1:02 pm</li>
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<li>Liquid culture checked @ 1:58 pm</li>
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<li>ESA Plated Culture: Checked at 1:59 pm</li>
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<li>-Sender Plate (was improvised) and Checked at 2:02 pm</li>
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<li>Liquid culture checked @ 2:58 pm</li>
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<li>ESA Plated Culture: Checked at 2:59 pm - Induction has been started (minimum 4-5 hrs for ESAI) </li>
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<li>-Sender Plate (was improvised) and Checked at 3:08 pm</li>
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<li>Liquid culture checked @ 3:58 pm</li>
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<li>ESA Plated Culture: Checked at 3:59 pm</li>
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<li>-Sender Plate (was improvised) and Checked at 4:08 pm</li>
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</ul>
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<p>(initial plates need to be analyzed from an 18hr incubation time and distance migrated)</p>
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<h3> Thursday, 7/20 </h3>
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<p>Plates were checked and expression was seen in all components and all of the cells that can produce GFP. Though the GFP+ is still lacking expression compared to the GFP+ only plate. The Plated Culture and the negative Sender plates had the greatest amount of GFP+ though this is not of much concern as it is a cell that is simply there to verify that fluorescence is able to be seen. Since the 2620 is well characterized and expresses in each run the lack of GFP+ expression hasn't been much of an issue. Another phenomena that was noticed was the variation in the machine's exposure. I was using set exposure settings in the manual capture mode along with the UV 6 and Epi UV setting. it seems that the camera is still auto exposing to the brightest emitted light. This means that the relative exposure of all samples is dependent on the brightest object being photographed. So If I only have the GFP+ in the frame then the machine captures more of its detail and shows a (possible) different picture than when the GFP+ is imaged next to the 2620 which is marginally brighter than the GFP+</p>
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<p>Today I will be investigating over time lapse the progression of GFP expression in the 2620. I will be plating ESAI as the sender at 1.5 inches away from the 2620 receiver as well as at .25 inches away from 2620 and placing them side by side under the imager if possible in order to time lapse both of their progressions. It is my aim to have a more uniform spread for this aspect of the experiment and I will be looking for time points of when expression begins and progresses towards its maximum. </p>
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<ul>
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<li>Plated ESAI at 1:55 pm</li>
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<li>Plated LuxI at 2:00 pm </li>
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<li>Putting in Incubator at 2:20 pm till I leave the lab which should be around 5 pm. </li>
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<li>There may be an issue with the plates as the 2620 is auto-inducing </li>
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<li>Because 2620 was auto-inducing (and LUX was already induced.. possible auto-inducing also) I took another image at 5:22 pm and left the plates in the imaging device in order to gather 50 images over the course of 12.5 hours to see if the LUX/ESA/2620 will have a trend of induction at room temperature. While this will not show us the change from non-induced to induced and beyond... It will show us a possible trend of induction from where they started at 5:22pm on 7-20-17</li>
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</ul>
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<h2>Induction Plates TraR, AubI, BjaI, CerI, LasI, LuxI, RpaI, EsaI</h2>
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<h3> Sunday, 10, 1</h3>
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These induction plates will have all senders with TraR (AubI, BjaI, CerI, LasI, LuxI, RpaI, TraI, EsaI) and one sender with LuxR (Bja).
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These plates will be setup the same as previous induction plate (GFP+, -Rec, Rec)
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<h3> Saturday, 10/7 </h3>
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<p>The induction plates were supposed to be setup on 10/1/17 but the TraR receiver, after transformation did not have enough viable colonies in order to make induction plates with 7 other receivers. Plates were setup on 10/6 and were left overnight to incubate at 37 degrees celsius since TraR is notorious for being slow to induce. </p>
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<p>Unfortunately after 24 hours of imaging (1st image was at 12 hours after incubation and last image was at 24 hrs) there was little to no evidence of induction on the plates. This may be due to either the the Receiver being 5 days old or that the senders were 5 days old. It seems that upon refrigeration and after extended periods of time on Amp agar, many of the cells lose the ability to send or possibly receive signals via AHL's. This might be due to the fact that when the cells multiply there is somatic recombination and the genes that control the AHL's production just get folded out. There is also the possibility that during the initial transformation of the TraR and related senders the intended insert was not taken up by the new cells. </p>
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  </table>
 
  </table>
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<p> After the digest was completed, I ran an agarose gel to confirm the digestion. The results are displayed below. I realized after running the gel that it would be very hard to confirm whether or not the digest was successful. This is because the size that I digested out is less than 100 nucleotide difference between the original PCR cleanup and the digested version. The next step I need to take is to gel extract the top band, ligate it and then transform it in DH5(alpha) turbo cells. </p>
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<center><img src="https://static.igem.org/mediawiki/2017/1/13/Bja_double_digest.png" alt="Design Flowchart" style="max-width: 400px; width: 70%"></center>
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<h1><ins> Notebook: Xylaan Livingstone </ins></h1>
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<h3>July 31st , 2017</h3>
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<h3><ins>Mini Prep for backbone:</ins></h3>
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<ul>
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<li>3 15 mL tubes of the LB Cor C</li>
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<li>3 15 mL tubes of LB AMP A3</li>
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</ul>
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<ol type="1">
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<li>Centrifuge down for pellet at highest speeds</li>
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<li>Each tube, I used 200 uL of resuspension fluid and transfer each to 2 mL tubes</li>
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<li>Each tube added lysis (200 uL), invert the tubes 5 times and immediately added 350 ul of neutralization buffer</li>
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<li>Cap each tube and centrifuge for 10 minutes at max speeds </li>
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<li>While centrifuging, I got 6 microfuge tubes for column prep, Put 6 columns in each pipet 500 uL of column prep into each</li>
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<li>Transfer the lysate from the 10-minute centrifuge to the labeled columns, then centrifuge the flow through </li>
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<li>Dump the flow through after </li>
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</ol>
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<p>~Proceeded with the same measurements from sigma mini prep</p>
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<p>~ Error with the elution step, after water flow through on the last step. 2 PJ1A3 tubes DNA elution was poured out on accident, so a lower concentration for two b was expected </p>
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<h3>Mini Preps Concentration</h3>
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<h4>1st Concentration results</h4>
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<table style="width:100%">
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  <tr>
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    <th>PSB1A3 </th>
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    <th>PSB1C3</th>
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  </tr>
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  <tr>
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    <td>16.068  ng/uL</td>
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    <td>6.024  ng/uL</td>
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  </tr>
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  <tr>
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    <td>41.607 ng/uL</td>
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    <td>40.571  ng/uL</td>
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  </tr>
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  <tr>
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    <td>12.99 ng/uL</td>
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    <td>7.578 ng/uL</td>
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  </tr>
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</table>
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<h2>August 1, 2017</h2>
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<p>Yesterday’s concentration created low DNA concentration. Thus, we are doing a second batch.</p>
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<p>2mL of PSB1C3 </p>
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<p>2mL of PS1A3 A3 </p>
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<ul>
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<li>All have fairly the same expression/ Approximately</li>
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<li>Bl21 bacteria for both batches </li>
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<li>Ran same mini prep procedure as yesterday, as well found in the Sigma mini prep procedure </li>
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<p>~Side note: it’s possible I am getting a lot of expression but the bacteria is not taking the plasmid </p>
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<h3>2nd Concentrations Results:</h3>
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<table style="width:100%">
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  <tr>
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    <th>PSB1A3</th>
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    <th>PSB1C3</th>
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  </tr>
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  <tr>
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    <td>16.158 ng/uL</td>
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    <td>31.964 ng/uL</td>
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  </tr>
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  <tr>
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    <td>37.435 ng/uL</td>
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    <td>14.273  ng/uL</td>
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  </tr>
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</table>
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<p>Will talk to graduate/ higher up to see a different result or possible error in steps of mini prep.</p>
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<h2>August 1st, 2017</h2>
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<p>Liquid Culture of </p>
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<ul>
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<li>3ml Cor C3</li>
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<li>3mL Amp A3</li>
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</ul>
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<p>New liquid cultures overnight, two different tubes.</p>
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<ul>
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<li>PSB1A3 in AMP</li>
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<li>PSB1C3 in Cor </li>
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<li>Left in the incubator at 3:21 p.m.</li>
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<li>Coming in at 10:00 a.m. to finish </li>
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</ul>
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<h2>August 2nd, 2017</h2>
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<center><img src=https://static.igem.org/mediawiki/2017/3/3b/Mcherry.png></center>
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<ul>
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<li>Took liquid culture out at 12:45 p.m. </li>
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<li>Ran mini-prep procedure. Instead of using neutralization in fridge, I am using a bottle at room temperature </li>
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<li>Lysis/ neutralization </li>
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<li>Accidently put 550 uL of lysis solution within tubes to counter balance mistake, I used 700 uL of neutralization solution to make up amount. </li>
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</ul>
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<h3> Concentration results: </h3>
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<table style="width:100%">
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  <tr>
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    <th>PSB1A3</th>
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    <th>PSB1C3</th>
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  </tr>
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  <tr>
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    <td>122.576 ng/uL</td>
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    <td>98.408 ng/uL</td>
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  </tr>
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</table>
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<p>Placed in freezer 2:20 P.M.</p>
  
  
 
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Latest revision as of 21:45, 28 October 2017

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