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 ran gel electrophoresis to yield the following gel -
• GEL PICTURE
• 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 -
• GEL PICTURE
• Mini-prepped them and confirmed the sequences.