Team:BostonU/FusionProteinsNotebook
From 2014.igem.org
(Difference between revisions)
(11 intermediate revisions not shown) | |||
Line 5: | Line 5: | ||
<div class="pagescontent"> | <div class="pagescontent"> | ||
- | |||
+ | <br><br> | ||
<pageheader>Notebook: Fusion Proteins</pageheader> | <pageheader>Notebook: Fusion Proteins</pageheader> | ||
Line 13: | Line 13: | ||
<table width="100%" border="0" cellspacing="15" cellpadding="0"> | <table width="100%" border="0" cellspacing="15" cellpadding="0"> | ||
<tr> | <tr> | ||
- | < | + | <td scope="col"> This notebook describes all the steps that were taken in constructing, and testing fusion proteins. The following ladder was used to determine the size of bands on all gel images in this notebook</td> </tr> |
<br><br> | <br><br> | ||
<tr> | <tr> | ||
- | < | + | <td scope="col"> |
- | <left><img src="https://static.igem.org/mediawiki/2014/9/9e/N3200_thumb.gif"></left></ | + | <left><img src="https://static.igem.org/mediawiki/2014/9/9e/N3200_thumb.gif"></left></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<br> | <br> | ||
- | < | + | <td colspan="2" scope="col"><br><h2>June</h2></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | < | + | <td colspan="2" scope="col"><h3>Week of June 23</h3></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | < | + | <td colspan="2" scope="col">Decided to make the following Level 0 Coding Sequences:<br> C0040_CI <br> |
C0080_CI <br> E0040m_ID <br> E0030_ID | C0080_CI <br> E0040m_ID <br> E0030_ID | ||
Line 44: | Line 44: | ||
<tr> | <tr> | ||
- | < | + | <td colspan="2" scope="col"><br><h3>Week of June 30</h3></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | < | + | <td 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. |
<ul> | <ul> | ||
<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. | <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. | ||
<li>The following transcriptional units will be next assembled so that the fusion proteins can be tested efficiently:<ol type= "I"> | <li>The following transcriptional units will be next assembled so that the fusion proteins can be tested efficiently:<ol type= "I"> | ||
- | <center><img src="https://static.igem.org/mediawiki/2014/f/f0/YABUwiki1.png" width = " | + | <center><img src="https://static.igem.org/mediawiki/2014/f/f0/YABUwiki1.png" width = "500" height = "100" alt="Pigeon img of a Fusion Protein" ></center> |
+ | <br> | ||
<li>J23100_AB - BCD2_BC - C0012_CD - B0015_DE | <li>J23100_AB - BCD2_BC - C0012_CD - B0015_DE | ||
<li>R0010_EB - BCD2_BC - C0040_CI - E0040m_ID - B0015_DF | <li>R0010_EB - BCD2_BC - C0040_CI - E0040m_ID - B0015_DF | ||
Line 71: | Line 72: | ||
<br> | <br> | ||
<tr> | <tr> | ||
- | < | + | <td colspan="2" scope="col"><br><h2>July</h2></td> |
</tr> | </tr> | ||
+ | </table> | ||
+ | <table width="100%" border="0" cellspacing="15" cellpadding="0"> | ||
- | <tr>< | + | <tr><td colspan="2" scope="col"><h3>Week of July 7</h3></td></tr> |
- | <tr>< | + | <tr><td colspan="2" scope="col"> This week I ran into problems with Kan plates, due to which I lost a lot of time. |
I then redid the transformations on new plates and could hence see blue and white colonies. | I then redid the transformations on new plates and could hence see blue and white colonies. | ||
<br> | <br> | ||
Line 91: | Line 94: | ||
<br> | <br> | ||
- | <tr>< | + | <tr><td colspan="2" scope="col"><h3>Week of July 14</h3></td></tr> |
- | <tr>< | + | <tr><td colspan="2" scope="col">This entire week I used the Google Glass for all my wet lab work. This was a part of a study run by the Human Computer Interaction Lab at Wellesley College. This week involved troubleshooting and making more of some Level 0 mini prep stocks were exhausted. |
<br> | <br> | ||
Line 116: | Line 119: | ||
</ul> | </ul> | ||
</tr> | </tr> | ||
- | <tr>< | + | <tr><td colspan="2" scope="col"><h3>Weeks of July 21 and July 28</h3></td></tr> |
- | <tr>< | + | <tr><td colspan="2" scope="col">Finally, I finished making the Level 1s. |
<br> | <br> | ||
Line 136: | Line 139: | ||
<br> | <br> | ||
<tr> | <tr> | ||
- | < | + | <td colspan="2" scope="col"><br><h2>August</h2></td> |
</tr> | </tr> | ||
- | <tr>< | + | <tr><td colspan="2" scope="col"><h3>Week of August 4</h3></td></tr> |
- | <tr>< | + | <tr><td colspan="2" scope="col">Troubleshooting for J00-C12m_AE and R10-C40-E40m_EF units |
<br> | <br> | ||
Line 161: | Line 164: | ||
- | <tr>< | + | <tr><td colspan="2" scope="col"><h3>Week of August 11</h3></td></tr> |
- | <tr>< | + | <tr><td colspan="2" scope="col">Testing WPI constructs and more cloning issues |
<br> | <br> | ||
Line 189: | Line 192: | ||
<br> | <br> | ||
</tr> | </tr> | ||
+ | </table> | ||
+ | <table width="100%" border="0" cellspacing="15" cellpadding="0"> | ||
+ | |||
- | <tr>< | + | <tr><td colspan="2" scope="col"><h3>Week of August 18 and August 25</h3></td></tr> |
- | <tr>< | + | <tr><td colspan="2" scope="col"> More FACS testing |
<br> | <br> | ||
Line 216: | Line 222: | ||
<tr> | <tr> | ||
- | < | + | <td colspan="2" scope="col"><br><h2>September</h2></td> |
</tr> | </tr> | ||
- | <tr>< | + | <tr><td colspan="2" scope="col"><h3>Week of September 1</h3></td></tr> |
- | <tr>< | + | <tr><td colspan="2" scope="col"> Started working on assembling Level 2s |
<br> | <br> | ||
Line 230: | Line 236: | ||
<center><img src="https://static.igem.org/mediawiki/2014/f/f4/FPYABUWiki.png" width="700" alt="Gel_8-31" style="float:center"></center> | <center><img src="https://static.igem.org/mediawiki/2014/f/f4/FPYABUWiki.png" width="700" alt="Gel_8-31" style="float:center"></center> | ||
- | <li>J00-C12_AE - R10-C40-E40m_EF - R40-E10_FG | + | <li>J00-C12_AE - R10-C40-E40m_EF - R40-E10_FG (A1) |
- | <li>J00-C12_AE - R10-C40-E30_EF - R40-E10_FG | + | <li>J00-C12_AE - R10-C40-E30_EF - R40-E10_FG (A2) |
- | <li>J00-C12_AE - R10-C80-E40m_EF - I13-E10_FG | + | <li>J00-C12_AE - R10-C80-E40m_EF - I13-E10_FG (A6) |
- | <li>J00-C12_AE - R10-C80-E30_EF - I13-E10_FG | + | <li>J00-C12_AE - R10-C80-E30_EF - I13-E10_FG (A7) |
<center><img src="https://static.igem.org/mediawiki/2014/3/3a/FP2YABUWiki.png" width="700" alt="Gel_8-31" style="float:center"></center> | <center><img src="https://static.igem.org/mediawiki/2014/3/3a/FP2YABUWiki.png" width="700" alt="Gel_8-31" style="float:center"></center> | ||
- | <li>J00-C12_AE - R10-C80_EF - I13-E10_FG | + | <li>J00-C12_AE - R10-C80_EF - I13-E10_FG (A8) |
- | <li>J00-C12_AE - R10-E30_EF - I13-E10_FG | + | <li>J00-C12_AE - R10-E30_EF - I13-E10_FG (A9) |
- | <li>J00-C12_AE - R10-E40m_EF - I13-E10_FG | + | <li>J00-C12_AE - R10-E40m_EF - I13-E10_FG (A10) |
- | <li>J00-C12_AE - R10-C40_EF - R40-E10_FG | + | <li>J00-C12_AE - R10-C40_EF - R40-E10_FG (A3) |
- | <li>J00-C12_AE - R10-E30_EF - R40-E10_FG | + | <li>J00-C12_AE - R10-E30_EF - R40-E10_FG (A4) |
- | <li>J00-C12_AE - R10-E40m_EF - R40-E10_FG</ol> | + | <li>J00-C12_AE - R10-E40m_EF - R40-E10_FG (A5)</ol> |
* All constructs contain BCD2_BC as 5' UTRs and B0015 as terminators. | * All constructs contain BCD2_BC as 5' UTRs and B0015 as terminators. | ||
Line 250: | Line 256: | ||
</tr> | </tr> | ||
- | <tr>< | + | <tr><td colspan="2" scope="col"><h3>Week of September 8</h3></td></tr> |
- | <tr>< | + | <tr><td colspan="2" scope="col"> Inter Lab Study |
<br> | <br> | ||
<ul> | <ul> | ||
Line 284: | Line 290: | ||
The gel didn't run well and none of the picked colonies had the right size. | The gel didn't run well and none of the picked colonies had the right size. | ||
</tr> | </tr> | ||
+ | </table> | ||
+ | <table width="100%" border="0" cellspacing="15" cellpadding="0"> | ||
- | <tr>< | + | <tr><td colspan="2" scope="col"><h3>Week of September 14 and 21</h3></td></tr> |
- | <tr>< | + | <tr><td colspan="2" scope="col"> Finished Inter-Lab study |
<br> | <br> | ||
<ul> | <ul> | ||
Line 302: | Line 310: | ||
<li> J23106 - E0240 (Cam) | <li> J23106 - E0240 (Cam) | ||
<li> J23115 - E0240 (Cam) | <li> J23115 - E0240 (Cam) | ||
- | <tr>< | + | <tr><td colspan="2" scope="col"> Resumed Phase 1 assembly |
<ul> | <ul> | ||
<li> Started building on Level 2s again using MoClo. | <li> Started building on Level 2s again using MoClo. | ||
+ | </tr> | ||
+ | |||
+ | <tr><td colspan="2" scope="col"><h3>Week of October 5 and 12</h3></td></tr> | ||
+ | |||
+ | <tr><td colspan="2" scope="col"> Phase 1 troubleshooting | ||
+ | <ul> | ||
+ | <li> Transformed MoClo reactions for the Level 2s and ran colony PCR | ||
+ | <br> | ||
+ | <center><img src="https://static.igem.org/mediawiki/2014/d/d3/YABUWiki1013-1.jpg" width="700" alt="Gel_8-31" style="float:center"></center> | ||
+ | <center><img src="https://static.igem.org/mediawiki/2014/1/11/YABUWiki1013-2.jpg" width="700" alt="Gel_8-31" style="float:center"></center> | ||
+ | <capt><br><center> Atleast one copy of A3, A4, A5, A9 and A10 have a band of the right size. The problem here is that many wells seem to have multiple bands of a range of sizes that are consistent across the board. This may have been a result of contamination. (See the Week of September 1 for description of Ax labels)</center></capt> | ||
+ | <br> | ||
+ | <li> Seeing that there were at least some bands of the right size, we decided to use that to our advantage and rerun colony PCR for A3, A4, A5, A9 and A10 using the stab plate made when I ran the first one. | ||
+ | <li> Then, I cut out just the bands that had the right size and proceeded to ligate them into pSB1C3 backbones. | ||
+ | <li> I couldn't finish transforming the Level 2s that worked in time for the Wiki freeze deadline. However, we should be done with this before the Giant Jamboree and present our results. | ||
+ | |||
</tr> | </tr> | ||
Latest revision as of 03:11, 16 October 2014
This notebook describes all the steps that were taken in constructing, and testing fusion proteins. The following ladder was used to determine the size of bands on all gel images in this notebook | |
|
|
June |
|
Week of June 23 |
|
Decided to make the following Level 0 Coding Sequences: C0040_CI C0080_CI E0040m_ID E0030_ID
| |
Week of June 30 |
|
This week was about sequence verifying the genes made earlier and laying down the plan for final testing of the fusion protein.
| |
July |
Week of July 7 | |
This week I ran into problems with Kan plates, due to which I lost a lot of time.
I then redid the transformations on new plates and could hence see blue and white colonies.
Poured LB, LB+Amp, and LB+Kan plates | |
Week of July 14 | |
This entire week I used the Google Glass for all my wet lab work. This was a part of a study run by the Human Computer Interaction Lab at Wellesley College. This week involved troubleshooting and making more of some Level 0 mini prep stocks were exhausted.
| |
Weeks of July 21 and July 28 | |
Finally, I finished making the Level 1s.
| |
August |
|
Week of August 4 | |
Troubleshooting for J00-C12m_AE and R10-C40-E40m_EF units
| |
Week of August 11 | |
Testing WPI constructs and more cloning issues
|
Week of August 18 and August 25 | |
More FACS testing
| |
September |
|
Week of September 1 | |
Started working on assembling Level 2s
| |
Week of September 8 | |
Inter Lab Study
|
Week of September 14 and 21 | |||||||
Finished Inter-Lab study
|