Team:BostonU/FusionProteins

From 2014.igem.org

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<th colspan="2" scope="col">BM3RI, BetI, Ph1F, SrpR, LmrA, L3S2P21, ECK120015170
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<th colspan="2" scope="col">This week was about sequence verifying the genes made earlier and laying down the plan for final testing of the fusion protein.
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&nbsp;&nbsp;&nbsp; <ul><li>Ran gel for previous PCR reactions <ol type= "I"><li>Two terminators were too small to see well on gel</ol> <li>PCR in triplicate for all genes and terminators as well as negative control <li>PCR clean-up <ol type= "I"> <li>Didn't work for ECK120015170 because it was too small & fell through the filter </ol> <li>Level 0 MoClo reactions for BM3RI, BetI, SrpR, LmrA, L3S2P21 <li>Redid PCR for ECK120015170 <li>Level 0 MoClo reaction for ECK120015170 <li>Transformations for all Level 0 MoClo reactions <ol type="I"> <li>None of the transformations worked <li>Accidentally used Pro cell strain with Cam resistance </ol> <li>Redid MoClo reactions and transformations for all parts <li>Ordered promoters for pBad, pTet, pA1LacO, and R0051 <li>Used colony PCR to verify transformations worked </ul> </tr>
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&nbsp;&nbsp;&nbsp; <ul>
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<li>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.
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<li>The following transcriptional units will be next assembled so that the fusion proteins can be tested efficiently:<ol type= "I">
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<li>J23100_AB - BCD2_BC - C0012_CD - B0015_DE
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<li>R0010_EB - BCD2_BC - C0040_CI - E0040_ID - B0015_DF
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<li>R0010_EB - BCD2_BC - C0040_CI - E0030_ID - B0015_DF
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<li>R0010_EB - BCD2_BC - C0080_CI - E0040_ID - B0015_DF
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<li>R0010_EB - BCD2_BC - C0080_CI - E0030_ID - B0015_DF
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<li>R0010_EB - BCD2_BC - C0080_CD - B0015_DF
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<li>R0010_EB - BCD2_BC - C0040_CD - B0015_DF
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<li>R0010_EB - BCD2_BC - E0040_CD - B0015_DF
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<li>R0010_EB - BCD2_BC - E0030_CD - B0015_DF
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<li>R0040_FB-BCD2_BC-E1010_CD-B0015_DG
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<li>I13453_FB-BCD2_BC-E1010_CD-B0015_DG</ol>  
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<li>Didn't have miniprep stocks for R0040_FB, B0015_DG and I13453_FB. So, streaked them out.
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<li>Picked colonies and grew overnight cultures.
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<li>Made minipreps and quantified for the three parts that were streaked</ul> </tr>
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Revision as of 22:30, 18 July 2014



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 used the google glass for all protocols to give Wellesley College feedback on one of their software projects.
BM3RI, BetI, Ph1F, SrpR, LmrA, L3S2P21, ECK120015170    
  • Ran gel for colony PCR reactions
  • Picked colonies to grow overnight for the parts that turned out well on the gel
    1. BM3RI, BetI, SrpR, LmrA, ECK120015170
  • Redid transformations for Ph1F, L3S2P21, SrpR, and ECK120015170
    1. SrpR and ECK120015170 looked questionable on the gel so I redid them just in case
  • Miniprepped overnight cultures and sent for sequencing
  • Analyzed sequencing results and confirmed BM3R1, BetI, LmrA, and ECK120015170
    1. SrpR came back as LacZ, which means it wasn't properly transformed
  • Picked confirmed colonies from stab plate, grew overnight, and made frozen stocks
  • Miniprepped SrpR, Ph1F, and L3S2P21 and sent for sequencing
  • Analyzed sequencing results and confirmed SrpR and L3S2P21
  • Redid colony PCR for Ph1F because it had too low concentration for sequencing

pBad, pTet, pA1LacO, R0051
  • Diluted pBad, pTet, pA1LacO, and R0051 primers
  • PCR for pBad, pTet, and pA1LacO
    1. Held off temporarily on R0051 because we didn't have the R0051_Rev_B primer that we thought we had
    2. For each part we made AK and KB fusion site using the new fusion site, K, that we designed (ATGC)
  • Level 0 MoClo reactions in DVL0_AB
    1. pTet-pBad, pTet-pA1LacO, pBad-pTet, pBad-pA1LacO, pA1LacO-pBad, pA1LacO-pTet
  • Transformations for level 0 tandem promoter MoClo reactions in bioline cells

Poured LB, LB+Amp, and LB+Kan plates

Week of July 14

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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