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:
    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 ran gel electrophoresis to yield the following gel -
    Gel showing the failure of parts with E0040


  • 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 PICTURE
  • Mini-prepped them and confirmed the sequences.







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