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Revision as of 08:54, 1 October 2016

Standard

Notebook.

In this page, you can view how we worked on our project.

Dairy

Week 1 (6/26/2016-7/02/2016)

Week 2 (7/03/2016-7/09/2016)

  • Parts Construction:
  • Protein Secretion:
    • Sequenced the plasmid pBES and confirmed that there are no additional BsaI sites
    • Transformed the plasmid pSB1C3 and extracted it for future use
    • Constructed the signal peptide ImdA on pSB1C3 vector

Week 3 (7/10/2016-7/16/2016)

  • Parts Construction:
  • Protein Secretion:
    • Constructed the signal peptide OmpA, LTIIb, PhoA, YjfA, NprE
  • Protein Purification:
  • Uranyl Absorption:
    • Pre-experiment:We got familiar with the apparatuses in the lab we performed experiments related to uranyl and tried the Arsenazo III method. Finally, we obtained a standard curve of the concentration ranging from 0uM-30uM

Week 4 (7/17/2016-7/23/2016)

  • Parts Construction:
  • Protein Secretion:
    • Constructed the signal peptide LipA, SacB
    • Constructed the kil protein
    • Tried to construct the OmpA-SUP-pET28a expression plasmid but failed
  • Protein Purification:
  • Retrivability:
    • We broke the E.coli with ultrasonication, and extracted 3A-mSA protein using a affinity column. Mixed 3A-mSA and 3B in TBS buffer. Then we did a SDS-PAGE electrophoresis and analysis the results ( Fig.1 ).We could find out that 3A-mSA had the ability to crosslink with 3B.

Week 5 (7/24/2016-7/30/2016)

  • Parts Construction:
  • Protein Secretion:
    • Constructed the expression plasmids OmpA-SUP-pET28a and LTIIb-SUP-pET28a and transformed them into BL21(DE3) strain
  • Protein Purification:
  • Retrivability:
    • We tried to measure the reaction capacity of the mSA part of the fusion protein with Biotin-Atto 488 which could show autofluorescence. But results were not useful, because there might be some noncovalent interactions between 3 kDa cutoff centrifuge filters and the molecular.

Week 6 (7/31/2016-8/06/2016)

  • Parts Construction:
  • Protein Secretion:
    • Constructed additional three signal peptides Bpr, Epr and PelB
    • Tried to construct the expression vector in B.subtilis but all failed
  • Protein Purification:
  • Uranyl Absorption:
    • We improved our experimental methods and supplemented equipment we needed. In these two days, we reduced our reaction volume from 3mL to 400uL and increased our efficiency. Also, standard curve of concentration ranging from 0uM-30uM was tested.
    • The adsorption capacity of 6A-SUP was tested, even though the data were not parallel, we confirmed that 6A-SUP can absorb uranyl. The highest adsorption rate was 81.56%
  • Retrivability:
    • Last week we got nothing useful. So we changed the protocol about how to use Biotin-Atto 488. We prepared several little affinity columns, immobilized 3A-mSA containing 6x His-Tag, and perfused the columns with solution of Biotin-Atto 488. After measuring the fluorescence intensity, we got a qualitative result that 3A-mSA can react with biotin.

Week 7 (8/07/2016-8/13/2016)

  • Parts Construction:
  • Protein Secretion:
    • Constructed the expression plasmids of SUP, mSA with different signal peptides: PhoA-SUP-pET28a, PelB-SUP-pET28a, OmpA-mSA-pET28a, LTIIb-mSA-pET28a, PhoA-mSA-pET28a and PelB-mSA-pET28a
    • Constructed the expression plasmids of kil: J23110-J61110-kil-pSB4C5
    • Constructed the expression plasmids of mRFP (E1010), yellow (K592010), eforRed (K592012): OmpA-mRFP-pET28a, LTIIb-mRFP-pET28a, PhoA-mRFP-pET28a, PelB-mRFP-pET28a, OmpA-Yellow-pET28a, LTIIb-Yellow-pET28a, PhoA-Yellow-pET28a, PelB-Yellow-pET28a, OmpA-eforRed-pET28a, LTIIb-eforRed-pET28a, PhoA-eforRed-pET28a and PelB-eforRed-pET28a
  • Protein Purification:
  • Uranyl Absorption:
    • The adsorption capacity of 3A-SUP and 3A-SUP+3B was tested. We confirmed that 3A-SUP can absorb uranyl, so can 3A-SUP+3B. In today’s experiments, the data was more parallel. We used solution containing uranyl only as control and its uranyl concentration after filtration as the actual concentration of uranyl.
  • Retrivability:
    • To absorb the proteins in the environment, we wanted to use biotin-coated beads. So we bought the reagents and magnetic beads with amino group.

Week 8 (8/14/2016-8/24/2016)

  • Protein Secretion:
    • Constructed the expression plasmids of SUP and Spycatcher of different signal peptides for B.subtilis: ImdA-SUP-pBES, NprE-SUP-pBES, SacB-SUP-pBES, YjfA-SUP-pBES, LipA-SUP-pBES, ImdA-Spycatcher-pBES, NprE-Spycatcher-pBES, SacB-Spycatcher-pBES, YjfA-Spycatcher-pBES, LipA-Spycatcher-pBES, Bpr-Spycatcher-pBES
  • Protein Purification:
  • Uranyl Absorption:
    • The adsorption capacity of His-SUP was tested, we confirmed that His-SUP can absorb uranyl and the result was close to the data performed by the author of the paper we referred.
    • We tested the adsorption capacity of 3/4/6A-SUP and 3/4/6A-SUP+3B in one day to exclude unnecessary effects. Then we analyzed the data and found the results were compromising. The capacity were fair and even though the proteins formed colloid, the adsorption capacity barely decreased.
  • Retrivability:
    • Constructed the biotin-coated beads.

Week 9 (8/21/2015-8/27/2015)

  • Parts Construction:
  • Protein Secretion:
    • BBa_J33201 (arsR)
    • Plasmids were isolated using a miniprep kit.
  • Protein Purification:
    • BBa_J33201 (arsR)
    • Plasmids were isolated using a miniprep kit.
  • Uranyl Absorption:
    • Water from Weiming lake was collected and simulated sea water was prepared.
    • We tested the adsorption capacity of 3A-SUP+3B in different conditions, including TBS buffer, Weiming lake and simulated sea water. Besides, we changed the protein-uranyl ratio from 1:1 to 10:1 to determine whether the adsorption capacity increased. Next, we decreased the uranyl concentration to 5uM. What’s more, we decreased the uranyl concentration to 13nM and increased the protein-uranyl ratio to 6000:1
  • Retrivability:
    • Prepared the solution containing 3A protein or 3A-mSA protein, added the beads we made last week, shocked the reaction system adequately for 1h, precipitated the beads with magnetic shelf, and measured the concentration of proteins in the liquid supernatant. We can get the quantitative data as Fig.2 shown.

Protocols

Gel Extraction

Ligation

Temperature Gradient Experiment

PCR

Purification of Recombinant Proteins

Transformation

Testing Adsorption Capacity of Protein

Agarose Gel Electrophoresis

Biotin-associated Experiment

SDS-Page

Western Blot

pH Gradient Experiment