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 (A1)
  2. J00-C12_AE - R10-C40-E30_EF - R40-E10_FG (A2)
  3. J00-C12_AE - R10-C80-E40m_EF - I13-E10_FG (A6)
  4. J00-C12_AE - R10-C80-E30_EF - I13-E10_FG (A7)
  5. J00-C12_AE - R10-C80_EF - I13-E10_FG (A8)
  6. J00-C12_AE - R10-E30_EF - I13-E10_FG (A9)
  7. J00-C12_AE - R10-E40m_EF - I13-E10_FG (A10)
  8. J00-C12_AE - R10-C40_EF - R40-E10_FG (A3)
  9. J00-C12_AE - R10-E30_EF - R40-E10_FG (A4)
  10. J00-C12_AE - R10-E40m_EF - R40-E10_FG (A5)
  * 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
  

Week of September 8

• Got the following plasmids passed on to me by Ariella -
  1. J23100-E0240 (Amp) - required
  2. J23101-E0240 (Amp) - required
  3. J23101-E0240 (Kan) - required
  4. J23103-E0240 (Amp)
  5. J23104-E0240 (Amp)
  6. J23107-E0240 (Amp)
  7. J23110-E0240 (Amp)
  8. J23112-E0240 (Amp)
  9. J23113-E0240 (Amp)
  10. J23116-E0240 (Amp)
  11. J23118-E0240 (Amp)
  12. J23119-E0240 (Amp)
• Ran overnight digests on inserts and necessary Cam backbones
  Bands clearly have the right size
• Cut out the bands and extracted out the plasmids
  
• Ligated the inserts to the appropriate backbones overnight and transformed them
• The following inserts transformed well -
  1. J23100-E0240 (Cam) - required
  2. J23101-E0240 (Cam) - required
  3. J23103-E0240 (Cam)
  4. J23113-E0240 (Cam)
  5. J23119-E0240 (Cam)
• Miniprepped the above plasmids.
• Also transformed and ran colony PCR on all the Level 2s. The gel didn't run well and none of the picked colonies had the right size.

Week of September 14 and 21

• Sequence confirmed the Inter-lab study constructs that transformed well
• Tested the required constructs on the flow cytometer
• Redid digests and ligations for the constructs that didn't transformed well
• After taking permission from the Measurement Track judges for it, I decided to streak out the following MoClo constructs for the study -
  1. J23108- E0240 (Kan)
  2. J23109- E0240 (Kan)
  3. J23111- E0240 (Kan)
• Traci worked on the following constructs -
  1. J23105 - E0240 (Cam)
  2. J23106 - E0240 (Cam)
  3. J23115 - E0240 (Cam)

• Started building on Level 2s again using MoClo.

Week of October 5 and 12

• Transformed MoClo reactions for the Level 2s and ran colony PCR
  
  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)
  
• 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.
• Then, I cut out just the bands that had the right size and proceeded to ligate them into pSB1C3 backbones.
• 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.