Team:BostonU/FusionProteins

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             <th scope="col"> 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. </th> </tr>
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             <th scope="col"> <Notebook Overview> </th> </tr>
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Revision as of 21:42, 18 July 2014



Notebook: Fusion Proteins



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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