Team:BostonU/FusionProteinsNotebook

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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: J23100_AB - BCD2_BC - C0012_CD - B0015_DE R0010_EB - BCD2_BC - C0040_CI - E0040m_ID - B0015_DF R0010_EB - BCD2_BC - C0040_CI - E0030_ID - B0015_DF R0010_EB - BCD2_BC - C0080_CI - E0040m_ID - B0015_DF R0010_EB - BCD2_BC - C0080_CI - E0030_ID - B0015_DF R0010_EB - BCD2_BC - C0080_CD - B0015_DF R0010_EB - BCD2_BC - C0040_CD - B0015_DF R0010_EB - BCD2_BC - E0040m_CD - B0015_DF R0010_EB - BCD2_BC - E0030_CD - B0015_DF R0040_FB-BCD2_BC-E1010_CD-B0015_DG 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 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 - J23100_AB - BCD2_BC - C0012_CD - B0015_DE R0010_EB - BCD2_BC - C0040_CI - E0030_ID - B0015_DF R0010_EB - BCD2_BC - C0080_CI - E0030_ID - B0015_DF R0010_EB - BCD2_BC - C0080_CD - B0015_DF R0010_EB - BCD2_BC - C0040_CD - B0015_DF R0010_EB - BCD2_BC - E0030_CD - B0015_DF R0040_FB-BCD2_BC-E1010_CD-B0015_DG 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
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 - 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 and more cloning issues 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
More FACS testing 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 Volunteered to take over and finish the InterLab Study.
Week of August 24