Team:Tianjin/Note/CFPS

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Notes For CFPS system

Week1(8/24/2016-8/30/2016)

Ⅰ.Constraction of expression plasmid in CFPS(cell-free protein synthesis)
The mutants of PETase gene had been contracted by over-lap PCR and Restriction Enzyme cutting sites(SacⅠ&EcoRⅠ) had been added to it’s ends (5’&3’) separately.

  • Transformation of E.coli (Trans-T1 Phage Resistant Chemically Competent Cell) with pRset_CFP-1(with AmpR) and plating on solid LB culture medium with Amp.
  • Cultivation the E.coli cells transformed yesterday in several 5mL liquid LB culture mediums with Amp at 37℃ for 12 hours.
  • Plasmid isolation of pRset_CFP-1.
  • Primers were designed for the target (CFP gene) within the plasmid pRset_CFP-1.
  • PCR with Q5-Polymerase to amplify CFP gene out of plasmid pRset_CFP-1 and add Restriction Enzyme cutting sites(XbaⅠ&SacⅠ) to it’s ends(5’&3’) separately.
    Bands as expected(about 760bp) in agarose gel , gel purification of the PCR products.

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    Week2(8/31/2016-9/6/2016)

  • XbaⅠ&SacⅠdouble restriction endonuclease digestion in CFP gene.

  • XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1
    Bands as expected(760bp&3000bp) in agarose gel , gel purification of the digested products(3000bp).

  • Ligaion of digested pRset_CFP-1, digested CFP gene and digested PETase(M154L) gene in accordance with the 1:5:5 molecular ratio. The newly constructed plasmid is called pRset_CFP-1-M154L.
    Transformation of E.coli (Trans-T1 Phage Resistant Chemically Competent Cell) with pRset_CFP-1-M154L(with AmpR) and plating on solid LB culture medium with Amp.
    Verification : Colony PCR with fast taq Polymerase, primers: PETase-S/PETase -A, template: Colony of E.coli with plasmid pRset_CFP-1-M154L. Bands as expected(about 760bp) in agarose gel.

    • T--Tianjin--cell-free_note-1
  • Cultivation the E.coli cells transformed yesterday in several 5mL liquid LB culture mediums with Amp at 37℃ for 12 hours.

  • Plasmid isolation of pRset_CFP-1-M154L.

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    Week3(9/7/2016-9/13/2016)

  • Our strategy is to exchange the site-directed mutant PETase-M154L for the other 21 mutants and 1 wild type, separately.
    XbaⅠ&EcoRⅠdouble restriction endonuclease digestion in pRset_CFP-1-M154L
    No expected bands(3600bp&750bp) in agarose gel. We don’t find the reason why the plasmid constuced can’t be cut into the two expected bands, so we decided to change the initial strategy.

  • Ligaion of digested pRset_CFP-1, digested CFP gene and the rest 22 digested gene interested (including 21 PETase site-directed mutants geneand 1 wild type ) separately in accordance with the 1:5:5 molecular ratio. The newly constructed plasmid is called pRset_CFP-1-R90I (R90T, R90A, S92A, Q119A, M154A, W159H, W159M, N205L, N205V, S207T, S207A, I208V, S238F, S238V,A240P, S242I.)

  • Transformation
  • Plasmid isolation
  • Verification

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    Week4(9/14/2016-9/20/2016)

    Ⅱ. Expression of the plasmids constracted before
  • We choosed 4 tubes of plasmid to express in the cell-free system firstly.
    The protocol of the cell-free protein synthesis system(50 µL) we used :
    MQ H2O     7.9µL
    Feeding buffer    25µL
    Mg2+ solution    1.1µL
    Gene( plasmid as template)  1µL
    Lysate     15µL
    (PS: The details of the system are not available.)
    • Systems were expressed in 96-well plates and put the 96-well plates into the ELIASA (microplate reader) at 30℃ for 12 hours.
    T--Tianjin--cell-free_note-2
  • Parallel experiments for expression in CFPS system

    • T--Tianjin--cf-blank T--Tianjin--cf-m1 T--Tianjin--cf-m2 T--Tianjin--cf-m3 T--Tianjin--cf-m4 T--Tianjin--cf-m5 T--Tianjin--cf-m6 T--Tianjin--cf-m7 T--Tianjin--cf-m8 T--Tianjin--cf-m9 T--Tianjin--cf-m10 T--Tianjin--cf-m11 T--Tianjin--cf-m12 T--Tianjin--cf-m13 T--Tianjin--cf-m14 T--Tianjin--cf-m15 T--Tianjin--cf-m16 T--Tianjin--cf-m17 T--Tianjin--cf-m18 T--Tianjin--cf-m19 T--Tianjin--cf-m20 T--Tianjin--cf-m21 T--Tianjin--cf-m22 T--Tianjin--cf-m-no
    The figures above show that the plasmids can express in the CFPS system, although the results of the parallel experimennts indicate that the CFPS system is unstable.
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    Week5(9/21/2016-9/27/2016)

    Ⅲ.Detection of Enzyme Activity

  • Substrate: 1mM pNPA Acetonitrile solution.
    10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.
    This attempt of detection failed because our enzyme precipitated in acetonitrile.
  • Subsrtrate: 1mM pNPA Tris-HCl buffer.
    10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.
    This attempt of detection failed, too. Because we found that Tris can react with pNPA , and pNPA would be transfered to pNP, too.

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    Week6(9/28/2016-10/2/2016)

  • Substrate:0.2mM pNPA water solution.
    10 times diluted Enzyme solution(unpurified) mixed with the equal volume substrate.
    After static reaction at 39℃ for 8 hours, we detected the characteristic adsorption peak of the product ,pNP, which has no other characteristic adsorption peak except in 400nm.
    • T--Tianjin--cf-m-no The control group in the image above indicate that something else in the CFPS system can cause characteristic adsorption peak except in 400nm with the sunstrate.


  • Substrate:PET.
    PET was been put into 20 times diluted Enzyme solution(unpurified).
    After static reaction at 39℃ for 5 days, we detected the characteristic adsorption peak of the product ,MHET, which has no other characteristic adsorption peak except in 260nm. Besides, we also deteected the product by Multispectral Scanning.

    • Please visit our Result part for more detailed result.
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    Notice: This page is currently under construction. Contents in this page are temporaory and will be modified several times before the final release.     — 2016 iGEM Team Tianjin

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