Team:BostonU/FusionProteins

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Notebook: Fusion Proteins


This notebook will delineate the steps taken and the parts used to create a library of MoClo level 0 (AK, KB, AB fusion sites) tandem promoter parts as well as the level 1 and level 2 parts created to test them. It also includes the steps taken to add several new gene (CD fusion sites) and terminator (DF fusion sites) MoClo parts to our library.

June

Week of June 23
Plasmids from addgene:
   Genes: Bm3RI, BetI, Ph1F, SrpR, LrmA
   Terminators: L3S2P21, ECK120015170
   Destination Vectors: pICH89921, pICH82113    
  • Struck out genes and terminators onto Amp plates
  • Struck out Destination Vectors onto Kan plates
  • Picked two colonies from each plate- one for minipreps & one for frozen stocks
  • Ordered primers for genes and terminators
  • Miniprepped terminators and genes
  • Made frozen stocks for all plasmids
  • Diluted primers and did PCR reactions for genes and terminators

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

Week of June 30
BM3RI, BetI, Ph1F, SrpR, LmrA, L3S2P21, ECK120015170    
  • Ran gel for previous PCR reactions
    1. Two terminators were too small to see well on gel
  • PCR in triplicate for all genes and terminators as well as negative control
  • PCR clean-up
    1. Didn't work for ECK120015170 because it was too small & fell through the filter
  • Level 0 MoClo reactions for BM3RI, BetI, SrpR, LmrA, L3S2P21
  • Redid PCR for ECK120015170
  • Level 0 MoClo reaction for ECK120015170
  • Transformations for all Level 0 MoClo reactions
    1. None of the transformations worked
    2. Accidentally used Pro cell strain with Cam resistance
  • Redid MoClo reactions and transformations for all parts
  • Ordered promoters for pBad, pTet, pA1LacO, and R0051
  • Used colony PCR to verify transformations worked

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

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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