Difference between revisions of "Team:Tianjin/Note/CFPS"

Line 204: Line 204:
 
<li>We choosed 4 tubes of plasmid to express in the cell-free system firstly.<br/>
 
<li>We choosed 4 tubes of plasmid to express in the cell-free system firstly.<br/>
 
The protocol of the cell-free protein synthesis system(50 µL) we used :<br/>
 
The protocol of the cell-free protein synthesis system(50 µL) we used :<br/>
−
MQ H2O"                        "7.9µL<br/>
+
MQ H2O<"                        ">7.9µL<br/>
−
Feeding buffer"                "25µL<br/>
+
Feeding buffer<"                ">25µL<br/>
−
Mg2+ solution"                  "1.1µL<br/>
+
Mg2+ solution<"                  ">1.1µL<br/>
−
Gene( plasmid as template)"    "1µL<br/>
+
Gene( plasmid as template)<"    ">1µL<br/>
−
Lysate"                        "15µL<br/>
+
Lysate<"                        ">15µL<br/>
 
(PS: the details of the system are not available)<br/>
 
(PS: the details of the system are not available)<br/>
 
</li>
 
</li>

Revision as of 05:55, 30 September 2016

TEAM TIANJIN

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Team Tianjin-Attribution

Notes

Week1(8/1/2016-8/7/2016)

Constraction of expression plasmid in CFPS(cell-free protein synthesis)
The mutations 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(8/15/2016-8/21/2016)

  • 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.

  • 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.
    T--Tianjin--cell-free_note-1

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    • Week3(8/29/2016-9/4/2016)

  • Our strategy is to exchange the site-directed mutation PETase-M154L for the other 21 mutations 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 mutations 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(9/5/2016-9/11/2016)

  • 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
    Show More 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(9/12/2016-9/18/2016)

  • Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.
    • The sequencing results for both of them were error.
  • Colonies containing Ni inducible promoter were used to inoculate overnight cultures.
  • PCR was performed to check if the gene fragments were ligated correctly.
    • 13_ fwd and 15_rev on pT-13-19-15
    Gel electrophoresis showed that it failed.
  • Plasmids pCPC-3031-Ni were isolated using a miniprep kit.
  • Several PCRs were performed to check if the gene fragments were ligated correctly.
    • 1.13_fwd and 13_rev on pT-13-19-15
    2.19_fwd and 19_rev on pT-13-19-15
    3.15_fwd and 15_rev on pT-13-19-15
    4.13_fwd and 19_rev on pT-13-19-15
    5.19_fwd and 15_rev on pT-13-19-15
    6.13_ wd and 15_rev on pT-13-19-15
    The fourth and sixth ones were not successful.
  • Repetition: Phosphorylated 13 was ligated into pT-19-15 and transformed into E.coli via heat shock.
  • Repetition: Several PCRs were performed to check if the gene fragments were ligated correctly.
    • 1.13_fwd and 13_rev on pT-13-19-15
    2.13_fwd and 19_rev on pT-13-19-15
    The second one was failed.
    IMG_2956

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    Week6(9/19/2016-9/25/2016)

  • Medium preparation :BG-11.
  • 19_ fwd and 15_rev were used to amplify 19-15.
    • Gel electrophoresis showed that amplification of fragments was successfull.
  • Ligated 13 and 19-15 via overlap PCR.
    • This PCR worked well and the products were extracted from the gel using a Agarose Gel DNA Extration Kit.
  • Restriction digest on pCPC-3031-Ni with Sac I.
  • The fragment 13-19-15 was ligated onto T vector and transformed into E.coli via heat shock.
  • A colony PCR of pT-13-19-15 was performed with 12 colonies.
    • Two of these colonies containing pT-13-19-15 were used to inoculate overnight cultures.
  • 13-19-15 gene fragment was phosphorylated.
  • Phosphorylated 13-19-15 was ligated into pCPC-3031-Ni and transformed into E.coli via heat shock.
  • Plasmids pT-13-19-15 were isolated using a miniprep kit.
  • A colony PCR of pCPC-3031-Ni-13-19-15 was performed with 12 colonies.
    • Three colonies were proved to be true. And two of them were used to inoculate overnight cultures.
    IMG_2956

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