Team:BostonU/FusionProteinsNotebook

June

Week of June 23

• 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

• 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

• 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.

Week of July 14

• 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-
  
  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

• 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 -
  
  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.
  

August

Week of August 4

• 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

• 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
  
  
• I ran restriction digests with Bbs1 on both clones of the plasmid that I had and discovered lacZ fragments in them. This along with the presence of RFP and GFP in the J00-C12m_AE mini preps led to assume that our mini preps were contaminated and I proceeded to streak out all the parts again.
  

Week of August 18 and August 25

• Made more competent cells for late stage testing
• Tested the WPI constructs successfully and sent results to the iGEM team
• To fix the problem of mixed plasmids in the C0012m_CD mini prep, I transformed the plasmids and ran colony PCR on 20 colonies. The gel obtained was -
  
  Picked only one colony was sequencing because most of them looked to have the same and the right size (~1kb)
  
• Ran MoClo on both constructs again and ran colony PCRs on 10 colonies each.
  
  Picked only two colonies each for sequencing but kept others on a stab plate
  
• Sequence confirmed the two Level 1s and celebrated.
• Volunteered to take over and finish the InterLab Study.

September

Week of September 1

• Ran MoClo to assemble the following Level 2s -
  
  1. J00-C12_AE - R10-C40-E40m_EF - R40-E10_FG
  2. J00-C12_AE - R10-C40-E30_EF - R40-E10_FG
  3. J00-C12_AE - R10-C80-E40m_EF - I13-E10_FG
  4. J00-C12_AE - R10-C80-E30_EF - I13-E10_FG
  5. J00-C12_AE - R10-C80_EF - I13-E10_FG
  6. J00-C12_AE - R10-E30_EF - I13-E10_FG
  7. J00-C12_AE - R10-E40m_EF - I13-E10_FG
  8. J00-C12_AE - R10-C40_EF - R40-E10_FG
  9. J00-C12_AE - R10-E30_EF - R40-E10_FG
  10. J00-C12_AE - R10-E40m_EF - R40-E10_FG
  * All constructs contain BCD2_BC as 5' UTRs and B0015 as terminators.
  
• Also, made frozen glycerol stocks for J00-C12m_AE and R10-C40-E40m_EF