Team:BostonU/FusionProteins

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Notebook: Fusion Proteins


Notebook Overview

June

Week of June 23
Decided to make the following Level 0 Coding Sequences:
   C0040_CI
   C0080_CI
   E0040_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 - E0040_ID - B0015_DF
    3. R0010_EB - BCD2_BC - C0040_CI - E0030_ID - B0015_DF
    4. R0010_EB - BCD2_BC - C0080_CI - E0040_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 - E0040_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.
   
  • Ran gel for colony PCR reactions
  • 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

Wellesley. In addition to my wetlab work this week I cleaned the lab, refilled stocks, and autoclaved backup supplies. I also talked to female high school students about iGEM and my experience with science and research to encourage them to pursue a science related field in university.
pBad, pTet, pA1LacO
  • Created stab plate and picked two colonies from each transformation plate
  • Miniprepped overnight cultures and sent level 0 tandem promoter MoClo parts in for sequencing
  • Analyzed sequences
    1. Only the pTet-pBad tandem promoter turned out correctly
    2. Noticed that the pA1LacO promoter has a large repeating sequence
      1. PCR temperature that I used was too high (too specific causing reverse primer to bind to the wrong part)
  • Redid PCR for pBad (AK) and pA1LacO (AK, KB)

R0051
  • Received and diluted R0051_Rev_B primer

L3S2P21, SrpR
  • Made frozen stocks from the confirmed colonies







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