Team:BostonU/FusionProteinsNotebook

From 2014.igem.org

(Difference between revisions)
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<li> Ran colony PCRs for transformed reactions, and electrophoresis to yield the following gel-
<li> Ran colony PCRs for transformed reactions, and electrophoresis to yield the following gel-
<center><img src="https://static.igem.org/mediawiki/2014/a/a9/Week_of_July_14YABUWiki.tif"  alt="Gel showing the failure of the cloning of all transcriptional units with E0040_ID" ></center>
<center><img src="https://static.igem.org/mediawiki/2014/a/a9/Week_of_July_14YABUWiki.tif"  alt="Gel showing the failure of the cloning of all transcriptional units with E0040_ID" ></center>
-
<capt><br>None of the transcriptional units have the right size (1.6 kb)</capt>
+
<capt><br>None of the transcriptional units have the right size (1.5-2 kb)</capt>
<li> As clear from the picture, most of the Level 1 inserts weren't as big as expected (700 kb instead of 1.5-2 kb).
<li> As clear from the picture, most of the Level 1 inserts weren't as big as expected (700 kb instead of 1.5-2 kb).
We discovered that none of the Level 1s with E0040m_CD or E0040m_ID worked and when I went back to compare the sequence of the stock mini-prep with that of E0040m_CD, I found that we had been using E0040 instead of E0040m.  So, I streaked out E0040m_CD and remade E0040m_ID using Phusion PCR
We discovered that none of the Level 1s with E0040m_CD or E0040m_ID worked and when I went back to compare the sequence of the stock mini-prep with that of E0040m_CD, I found that we had been using E0040 instead of E0040m.  So, I streaked out E0040m_CD and remade E0040m_ID using Phusion PCR
Line 120: Line 120:
<br>
<br>
<li> Mini-prepped them and confirmed the sequences.  
<li> Mini-prepped them and confirmed the sequences.  
 +
<br>
 +
</tr>
 +
 +
<tr><th colspan="2" scope="col"><h3>Week of August 4</h3></th></tr>
 +
 +
<tr><th colspan="2" scope="col">Troubleshooting for J00-C12m_AE and R10-C40-E40m_EF units
 +
<br>
 +
 +
<ul><li> Tried re-cloning both constructs using more plasmid of C0012_CD for J00-C12_AE
 +
because Traci said that C0012_CD has 2 illegal sites that always causes problems when cloning it into L1s
 +
<li> When that didn't work, we found C0012m_CD in our MoClo library in which the sites were fixed.
 +
<li> We repeated MoClo for both constructs yet again and as evidenced by the gel below, the R10-C40-E40m_EF clone worked.
 +
 +
<center><img src="https://static.igem.org/mediawiki/2014/2/28/Week_of_August_4YABUWiki.tif"></center>
 +
<capt><br>Most of the clones on a gel look like they worked. J00-C12_AE is supposed to be 1.6 kb and R10-C40-E40m_EF is 2.2 kb</capt>
 +
<br>
 +
<li> Sequence confirmed all clones of R10-C40-E40m_EF but sequences for J00-C12m_AE weren't correct
 +
<li>Transformed all the existing fusion proteins and sequence confirmed transcriptional units to make glycerol stocks.
 +
<li> Also , transformed the plasmids for COXGR_AF, COXRG_AF, which are essential constructs for testing.
 +
Made glycerol stocks for them too.
 +
<br>
 +
</tr>
 +
 +
 +
<tr><th colspan="2" scope="col"><h3>Week of August 11</h3></th></tr>
 +
 +
<tr><th colspan="2" scope="col">Testing WPI constructs
 +
<br>
 +
 +
<ul>
 +
 +
<li>Volunteered to prepare collaboration constructs from WPI for testing and followed the FACS workflow protocol for it
 +
<li> Because FACS workflow was recently prepared, there was a lot of trial and error. I added too much of culture in the well plates, which then spilled over and gave faulty readings on the flow cytometer.
 +
<li> Transformed the C0012m_CD once again and before purifying the new plasmids noticed that the overnight cultures were green.
 +
<li>Went back to the transformation plate and observed red and green colonies along with a few white ones under UV light.
 +
<li>Screened all white colonies and that yielded the following gel-
 +
 +
<center><img src="https://static.igem.org/mediawiki/2014/7/79/0813.tif"></center>
 +
<capt><br>Only one clone seem to be of the right size.  J00-C12_AE is supposed to be 1.6 kb.</capt>
 +
<br>
 +
 +
<li>Transformed the sequence confirmed R10-C40-E40m_EF with XGAL and IPTG for blue-white screening. Curiously, I observed multiple blue colonies, which is very improbable for a plasmid transformation.
 +
<center><img src="https://static.igem.org/mediawiki/2014/b/b9/WeekofAug4YABUWiki.JPG"></center>
 +
<capt><br>More blue colonies than white ones. Shouldn't have happened for a plasmid transformation</capt>
 +
<br>
 +
<br>
<br>
</tr>
</tr>

Revision as of 04:15, 22 September 2014



Notebook: Fusion Proteins


The notebook below describes all the steps that were taken in constructing, and testing fusion proteins.

June

Week of June 23
Decided to make the following Level 0 Coding Sequences:
   C0040_CI
   C0080_CI
   E0040m_ID
   E0030_ID    
  • Used Phusion PCR to make the parts listed above and then, performed PCR cleanup to purify the DNA fragments.
  • 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.
  • Performed MoClo Level 0 reaction to insert the purified constructs into backbones with a Cam resistant gene.
  • Transformed the MoClo plasmids on Cam plates to perform Blue-White screening and pick colonies that had successful digestion-ligation
  • Transformation didn't work because I used faulty Bioline cells. So, I repeated transformations using DH5a cells.
  • Screened colonies further by performing Colony PCRs and running a gel.

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.    
  • 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.
  • The following transcriptional units will be next assembled so that the fusion proteins can be tested efficiently:
    1. J23100_AB - BCD2_BC - C0012_CD - B0015_DE
    2. R0010_EB - BCD2_BC - C0040_CI - E0040m_ID - B0015_DF
    3. R0010_EB - BCD2_BC - C0040_CI - E0030_ID - B0015_DF
    4. R0010_EB - BCD2_BC - C0080_CI - E0040m_ID - B0015_DF
    5. R0010_EB - BCD2_BC - C0080_CI - E0030_ID - B0015_DF
    6. R0010_EB - BCD2_BC - C0080_CD - B0015_DF
    7. R0010_EB - BCD2_BC - C0040_CD - B0015_DF
    8. R0010_EB - BCD2_BC - E0040m_CD - B0015_DF
    9. R0010_EB - BCD2_BC - E0030_CD - B0015_DF
    10. R0040_FB-BCD2_BC-E1010_CD-B0015_DG
    11. I13453_FB-BCD2_BC-E1010_CD-B0015_DG
  • Didn't have miniprep stocks for R0040_FB, B0015_DG and I13453_FB. So, streaked them out.
  • Picked colonies and grew overnight cultures.
  • Made minipreps and quantified for the three parts that were streaked

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.
   
  • After sequence verifying the mini preps of the Level 1s I made before, I found that the following Level 1s worked -
  • Setup the MoClo reactions for all the Transcriptional Units
  • Transformed all reactions on Kan plates
  • Plates did not have any growth. So, I repeated transformations another time and when that didn't work, repeated the MoClo reactions
    1. Finally, the transformations yielded blue and white colonies as expected.

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.
  • After sequence verifying the mini preps of the Level 1s I made before, I found that the following Level 1s worked -
    1. J23100_AB - BCD2_BC - C0012_CD - B0015_DE
    2. R0010_EB - BCD2_BC - C0040_CI - E0030_ID - B0015_DF
    3. R0010_EB - BCD2_BC - C0080_CI - E0030_ID - B0015_DF
    4. R0010_EB - BCD2_BC - C0080_CD - B0015_DF
    5. R0010_EB - BCD2_BC - C0040_CD - B0015_DF
    6. R0010_EB - BCD2_BC - E0030_CD - B0015_DF
    7. R0040_FB-BCD2_BC-E1010_CD-B0015_DG
    8. I13453_FB-BCD2_BC-E1010_CD-B0015_DG
  • Repeated all the MoClo for the all the Level 1s that didn't work.
  • Ran colony PCRs for transformed reactions, and electrophoresis to yield the following gel-
    Gel showing the failure of the cloning of all transcriptional units with E0040_ID

    None of the transcriptional units have the right size (1.5-2 kb)
  • As clear from the picture, most of the Level 1 inserts weren't as big as expected (700 kb instead of 1.5-2 kb). We discovered that none of the Level 1s with E0040m_CD or E0040m_ID worked and when I went back to compare the sequence of the stock mini-prep with that of E0040m_CD, I found that we had been using E0040 instead of E0040m. So, I streaked out E0040m_CD and remade E0040m_ID using Phusion PCR

Weeks of July 21 and July 28

Finally, I finished making the Level 1s.
  • Confirmed the E0040m_ID by sending it for sequencing.
  • Assembled the remaining Level 1s using Modular Cloning.
  • Transformed the MoClo reactions.
  • Performed colony PCRs on white colonies from all transformed reactions and ran the following gel -
    Gel showing the successful cloning of E0040m_ID

    One of the 3 clones of E0040m_ID was successful as evident by the size of the insert
  • Mini-prepped them and confirmed the sequences.

Week of August 4

Troubleshooting for J00-C12m_AE and R10-C40-E40m_EF units
  • Tried re-cloning both constructs using more plasmid of C0012_CD for J00-C12_AE because Traci said that C0012_CD has 2 illegal sites that always causes problems when cloning it into L1s
  • When that didn't work, we found C0012m_CD in our MoClo library in which the sites were fixed.
  • We repeated MoClo for both constructs yet again and as evidenced by the gel below, the R10-C40-E40m_EF clone worked.

    Most of the clones on a gel look like they worked. J00-C12_AE is supposed to be 1.6 kb and R10-C40-E40m_EF is 2.2 kb
  • Sequence confirmed all clones of R10-C40-E40m_EF but sequences for J00-C12m_AE weren't correct
  • Transformed all the existing fusion proteins and sequence confirmed transcriptional units to make glycerol stocks.
  • Also , transformed the plasmids for COXGR_AF, COXRG_AF, which are essential constructs for testing. Made glycerol stocks for them too.

Week of August 11

Testing WPI constructs
  • Volunteered to prepare collaboration constructs from WPI for testing and followed the FACS workflow protocol for it
  • Because FACS workflow was recently prepared, there was a lot of trial and error. I added too much of culture in the well plates, which then spilled over and gave faulty readings on the flow cytometer.
  • Transformed the C0012m_CD once again and before purifying the new plasmids noticed that the overnight cultures were green.
  • Went back to the transformation plate and observed red and green colonies along with a few white ones under UV light.
  • Screened all white colonies and that yielded the following gel-

    Only one clone seem to be of the right size. J00-C12_AE is supposed to be 1.6 kb.
  • Transformed the sequence confirmed R10-C40-E40m_EF with XGAL and IPTG for blue-white screening. Curiously, I observed multiple blue colonies, which is very improbable for a plasmid transformation.

    More blue colonies than white ones. Shouldn't have happened for a plasmid transformation








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