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| − | <h1>Notebook: Jimmy Xu</h1> | + | <h1>Notebook: Brianna Lopez</h1> |
| − | <h2>Wiki Design</h2> | + | <h2>Transformation of Negative Sender and LuxR</h2> |
| − | <h3>8/25 Thursday</h3> | + | <ul> |
| − | <p>This week, I added the menu bar to the Wiki, created the team logo, and designed the layout of the main page. I also added to the Project Description.</p> | + | <li>These colonies will be used for overnight liquid culture growth, which will be used in 96-well plating for overnight growth curves.</li> |
| − | <br> | + | <li>Transformation of a negative sender, and 2620 vector. </li> |
| − | <h3>9/1 Thursday</h3> | + | |
| − | <p>This week, I added to the Project Description, added 6 protocols to the Protocols tab, and created the Safety tab. I also edited the AubI and BjaI parts pages.</p> | + | <li>Concentration of 2620 & Neg Sender:</li> |
| − | <h3>9/8 Thursday</h3> | + | </ul> |
| − | <p>This week, I helped Jiaqi with psB1C3 cloning, doing a gel extraction on the digested plasmid. For the Wiki, I finished the Protocols tab, while also adding information to the About the Team tab. I also added information to the BraI and CerI parts pages. </p> | + | <p>-2620 = 135 ng/uL</p> |
| − | <h3>9/15 Thursday</h3> | + | <p>-Neg Sender = 461 ng/uL (will have to do 1:10 dilution)</p> |
| − | <p>This week, I helped Jiaqi with psB1C3 cloning, making liquid culture for Aub, Bja, Bra and Cer inserts. I also retransformed all 10 systems in BL21 for OD600 and mCherry test next week. For the Wiki, the Team description page was updated, as well as the Basic Parts, Composite Parts, and Part Collection tables. Also, I helped work on the presentation for a practice presentation next Monday.</p> | + | |
| − | <h3>9/22 Thursday</h3> | + | <ul> |
| − | <p>This week, I ran a 96-well plate over an 8-hour read to test OD600 and mCherry absorbance over time. This proof-of-concept experiment was done in order to demonstrate that it is likely that the AHL is being produced in E. coli. This is because the AHL gene is located upstream of the mCherry gene, so the production of mCherry should also mean AHL production. </p> | + | |
| − | <p>For the wiki, I worked on the Project Description, added Attributions, and more Safety information. The Human Practices page was also started.</p> | + | <li>Calculation Calculation to get concentration of 60 ng/uL each:</li> |
| − | <h3>9/29 Thursday</h3> | + | <li>Negative Sender: </li> |
| − | <p>This week, I retransformed the 10 systems and re-ran the OD tests over an 8 hour period, because the previous run had produced a negative trend of mCherry production over time. The results were more promising, but the Esa, Lux, Rhl, and Rpa systems will be rerun next week in order to produce reasonable data. </p> | + | </ul> |
| − | <p>For the wiki, I finished Attributions, worked on Human Practices and the Parts Overview tab, while also finishing up the Team description. I also added a graphic to the front page.</p> | + | <p>- 461 ng/ul (1uL into 9uL H20) = 46.1 ng/uL </p> |
| − | <h3>10/6 Thursday</h3> | + | <p>- 46.1 ng/uL * x uL = 60 ng </p> |
| − | <p>This week, I reran the OD test for the Esa, Lux, Rhl, and Rpa systems. These produced positive growth curves for mCherry over time, which is a good indicator of successful AHL production in E. coli.</p> | + | <p><ins>- x = 1.3 uL </ins></p> |
| − | <p>For the wiki, I set up the Sin and Modular Sender Vector parts pages. I also added to the Aub, Bja, Bra, and Cer parts pages. I added to the Project Description, and Parts Overview tabs.</p> | + | <ul> |
| − | <h3>10/13 Thursday</h3> | + | <li>2620: </li> |
| − | <p>This week, I added to the Parts Pages in the registry and finished the Attributions tab.</p> | + | </ul> |
| − | <h3>10/19 Wednesday</h3> | + | <p>- 135 ng/uL * x uL = 60 ng </p> |
| − | This week is the Wiki Freeze. I finished up the Wiki, with additions to Results, Parts Overview, Project Description, Notebook, and Human Practices.
| + | <p><ins> - x = .44 uL </ins></p> |
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| | + | <h3>Protocol:</h3> |
| | + | <p>Materials:</p> |
| | + | <ul> |
| | + | <li>Ice bucket </li> |
| | + | <li>2620 </li> |
| | + | <li>Negative Sender </li> |
| | + | <li>DI H20 </li> |
| | + | |
| | + | </ul> |
| | + | |
| | + | <h3>Dilution Procedure:</h3> |
| | + | <ol type="1"> |
| | + | <li>One 1.5mL tube was used to perform a dilution, and labeled dilution tube.</li> |
| | + | <li>9uL of DI H20 was pipetted into the dilution tube.</li> |
| | + | <li>1uL of negative sender was taken out of stock and pipetted into the dilution tube.</li> |
| | + | <li>The volume was then pipetted up and down to mix and dilute the solution. </li> |
| | + | </ol> |
| | + | |
| | + | <h3>Transformation Procedure:</h3> |
| | + | |
| | + | <ol type="1"> |
| | + | <li>One tube was labeled 2620, the other negative sender</li> |
| | + | <li>1.3uL of negative sender was pipetted from the dilution tube into the negative sender tube</li> |
| | + | <li>0.44uL of 2620 was pipetted from 2620 stock tube into the 2620 tube. </li> |
| | + | <li>Tubes were then placed on ice for 2-5 minutes</li> |
| | + | <li>The tubes were then placed on the hot plate of 42C for exactly 45 seconds then immediately placed on ice for 5 minutes. </li> |
| | + | <li>300uL of SOC was added to each tube </li> |
| | + | <li>It was then placed in the 37C shaking incubator for 20-30 minutes </li> |
| | + | <li>The tubes were then spun down at 3000 rpm for 1 minute </li> |
| | + | <li>The liquid in the tubes were gently tapped out into liquid waste</li> |
| | + | <li>100uL of SOC was added to the tubes and gently pipetted up and down to resuspend the cells</li> |
| | + | <li>100uL of each tube were then plated onto LB/AMP plates</li> |
| | + | <li>Then placed in 37̊C still incubator over night </li> |
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