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| - | <th scope="col"> Notebook Overview </th> </tr> | + | <th scope="col">Fusion proteins are analogous to tandem promoters. For our purposes, we fused multiple repressors with reporter proteins in order to check whether a transcriptional unit containing fusion proteins worked just as well as two transcriptional units (one with the repressor and the other with the reporter protein). These constructs were fused by adding unique fusion sites to their ends and then, ligating them using the <a href='https://2014.igem.org/Team:BostonU/MoClo'>Modular Cloning</a> method. The objective of making this library was to help us create standard transcriptional units that could universally be used to make intricate genetic circuits.</th> </tr> |
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| - | <th colspan="2" scope="col"><br><h2>June</h2></th>
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| - | <th colspan="2" scope="col"><h3>Week of June 23</h3></th>
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| - | </tr>
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| - | <th colspan="2" scope="col">Decided to make the following Level 0 Coding Sequences:<br> C0040_CI <br>
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| - | C0080_CI <br> E0040_ID <br> E0030_ID
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| - | <ul><li>Used Phusion PCR to make the parts listed above and then, performed PCR cleanup to purify the DNA fragments.
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| - | <li> Upon quantifying all clean-ups, I found that the concentration for the E0040_ID (4.4 ng/uL) cleanup was lower than that of the negative control (6.1 ng/uL). So, I repeated the Phusion PCR for that part and ended up with a concentration of 31.9 ng/uL.
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| - | <li> Performed MoClo Level 0 reaction to insert the purified constructs into backbones with a Cam resistant gene.
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| - | <li> Transformed the MoClo plasmids on Cam plates to perform Blue-White screening and pick colonies that had successful digestion-ligation
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| - | <li> Transformation didn't work because I used faulty Bioline cells. So, I repeated transformations using DH5a cells.
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| - | <li> Screened colonies further by performing Colony PCRs and running a gel.
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| - | </ul>
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| - | <th colspan="2" scope="col"><br><h3>Week of June 30</h3></th>
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| - | <th colspan="2" scope="col">This week was about sequence verifying the genes made earlier and laying down the plan for final testing of the fusion protein.
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| - | <li>Miniprepped the overnight cultures for the colonies with plasmids that contain the required insert and sent them in for sequencing. All the sequences were as expected.
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| - | <li>The following transcriptional units will be next assembled so that the fusion proteins can be tested efficiently:<ol type= "I">
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| - | <li>J23100_AB - BCD2_BC - C0012_CD - B0015_DE
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| - | <li>R0010_EB - BCD2_BC - C0040_CI - E0040_ID - B0015_DF
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| - | <li>R0010_EB - BCD2_BC - C0040_CI - E0030_ID - B0015_DF
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| - | <li>R0010_EB - BCD2_BC - C0080_CI - E0040_ID - B0015_DF
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| - | <li>R0010_EB - BCD2_BC - C0080_CI - E0030_ID - B0015_DF
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| - | <li>R0010_EB - BCD2_BC - C0080_CD - B0015_DF
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| - | <li>R0010_EB - BCD2_BC - C0040_CD - B0015_DF
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| - | <li>R0010_EB - BCD2_BC - E0040_CD - B0015_DF
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| - | <li>R0010_EB - BCD2_BC - E0030_CD - B0015_DF
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| - | <li>R0040_FB-BCD2_BC-E1010_CD-B0015_DG
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| - | <li>I13453_FB-BCD2_BC-E1010_CD-B0015_DG</ol>
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| - | <li>Didn't have miniprep stocks for R0040_FB, B0015_DG and I13453_FB. So, streaked them out.
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| - | <li>Picked colonies and grew overnight cultures.
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| - | <li>Made minipreps and quantified for the three parts that were streaked</ul> </tr>
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| - | <th colspan="2" scope="col"><br><h2>July</h2></th>
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| - | </tr>
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| - | <tr><th colspan="2" scope="col"><h3>Week of July 7</h3></th></tr>
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| - | <tr><th colspan="2" scope="col"> This week I ran into problems with Kan plates, due to which I lost a lot of time.
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| - | I then redid the transformations on new plates and could hence see blue and white colonies.
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| - | <ul><li>Ran gel for colony PCR reactions
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| - | <li>Setup the MoClo reactions for all the Transcriptional Units
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| - | <li> Transformed all reactions on Kan plates
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| - | <li> Plates did not have any growth. So, I repeated transformations another time and when that didn't work, repeated the MoClo reactions <ol type= "I">
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| - | <li> Finally, the transformations yielded blue and white colonies as expected. </ol>
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| - | </ul>
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| - | <br>
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| - | Poured LB, LB+Amp, and LB+Kan plates
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| - | </tr>
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| - | <tr><th colspan="2" scope="col"><h3>Week of July 14</h3></th></tr>
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| - | <tr><th colspan="2" scope="col">Wellesley. In addition to my wetlab work this week I cleaned the lab, refilled stocks, and autoclaved backup supplies. I also talked to female high school students about iGEM and my experience with science and research to encourage them to pursue a science related field in university.
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| - | <br>
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| - | pBad, pTet, pA1LacO
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| - | <ul><li>Created stab plate and picked two colonies from each transformation plate
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| - | <li>Miniprepped overnight cultures and sent level 0 tandem promoter MoClo parts in for sequencing
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| - | <li> Analyzed sequences <ol type= "I">
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| - | <li>Only the pTet-pBad tandem promoter turned out correctly
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| - | <li>Noticed that the pA1LacO promoter has a large repeating sequence <ol type="A">
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| - | <li> PCR temperature that I used was too high (too specific causing reverse primer to bind to the wrong part)</ol> </ol>
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| - | <li>Redid PCR for pBad (AK) and pA1LacO (AK, KB) </ul>
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| - | R0051
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| - | <ul><li>Received and diluted R0051_Rev_B primer </ul>
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| - | L3S2P21, SrpR
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| - | <ul><li>Made frozen stocks from the confirmed colonies</ul> </tr>
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| | </table> | | </table> |
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