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

Line 219: Line 219:
 
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/d/d0/T--Tianjin--cf-m1.png"  alt="T--Tianjin--cf-m1" width="800" height="533">
 
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/d/d0/T--Tianjin--cf-m1.png"  alt="T--Tianjin--cf-m1" width="800" height="533">
 
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m2.png"  alt="T--Tianjin--cf-m2" width="800" height="533">
 
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m2.png"  alt="T--Tianjin--cf-m2" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m2.png"  alt="T--Tianjin--cf-m2" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/3/34/T--Tianjin--cf-m3.png"  alt="T--Tianjin--cf-m3" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m3.png"  alt="T--Tianjin--cf-m3" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/7/75/T--Tianjin--cf-m4.png"  alt="T--Tianjin--cf-m4" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m4.png"  alt="T--Tianjin--cf-m4" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/b/be/T--Tianjin--cf-m5.png"  alt="T--Tianjin--cf-m5" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m5.png"  alt="T--Tianjin--cf-m5" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/4e/T--Tianjin--cf-m6.png"  alt="T--Tianjin--cf-m6" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m6.png"  alt="T--Tianjin--cf-m6" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/4e/T--Tianjin--cf-m7.png"  alt="T--Tianjin--cf-m7" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m7.png"  alt="T--Tianjin--cf-m7" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/9/97/T--Tianjin--cf-m8.png"  alt="T--Tianjin--cf-m8" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m8.png"  alt="T--Tianjin--cf-m8" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/d/d1/T--Tianjin--cf-m9.png"  alt="T--Tianjin--cf-m9" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m9.png"  alt="T--Tianjin--cf-m9" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/6/61/T--Tianjin--cf-m10.png"  alt="T--Tianjin--cf-m10" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m10.png"  alt="T--Tianjin--cf-m10" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/c/c9/T--Tianjin--cf-m11.png"  alt="T--Tianjin--cf-m11" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m11.png"  alt="T--Tianjin--cf-m11" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/3/36/T--Tianjin--cf-m12.png"  alt="T--Tianjin--cf-m12" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m12.png"  alt="T--Tianjin--cf-m12" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/d/d2/T--Tianjin--cf-m13.png"  alt="T--Tianjin--cf-m13" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m13.png"  alt="T--Tianjin--cf-m13" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/3/35/T--Tianjin--cf-m14.png"  alt="T--Tianjin--cf-m14" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m14.png"  alt="T--Tianjin--cf-m14" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/b/b3/T--Tianjin--cf-m15.png"  alt="T--Tianjin--cf-m15" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m15.png"  alt="T--Tianjin--cf-m15" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/2/2c/T--Tianjin--cf-m16.png"  alt="T--Tianjin--cf-m16" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m16.png"  alt="T--Tianjin--cf-m16" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/9/91/T--Tianjin--cf-m17.png"  alt="T--Tianjin--cf-m17" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m17.png"  alt="T--Tianjin--cf-m17" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/3/3f/T--Tianjin--cf-m18.png"  alt="T--Tianjin--cf-m18" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m18.png"  alt="T--Tianjin--cf-m18" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/a/ad/T--Tianjin--cf-m19.png"  alt="T--Tianjin--cf-m19" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m19.png"  alt="T--Tianjin--cf-m19" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/0/03/T--Tianjin--cf-m20.png"  alt="T--Tianjin--cf-m20" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m20.png"  alt="T--Tianjin--cf-m20" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/b/b9/T--Tianjin--cf-m21.png"  alt="T--Tianjin--cf-m21" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m21.png"  alt="T--Tianjin--cf-m21" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/3/3d/T--Tianjin--cf-m22.png"  alt="T--Tianjin--cf-m22" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m22.png"  alt="T--Tianjin--cf-m22" width="800" height="533">
+
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/3/3e/T--Tianjin--cf-m-no.png"  alt="T--Tianjin--cf-m-no" width="800" height="533">
−
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m-no.png"  alt="T--Tianjin--cf-m-no" width="800" height="533">
+
  
  

Revision as of 05:37, 30 September 2016

TEAM TIANJIN

LOGO


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.

    • 查看Team Tianjin全部实验 ›

    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

    查看Team Tianjin全部实验 ›

    • 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

    • 查看Team Tianjin全部实验 ›

    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
  • 13 amplification at 65.0°C with 13.rev/fwd primes
    • PCR worked, positive control worked, no amplification of 13
  • The fragments of 13 were purified with PCR Purification Kit.
  • 13 gene fragment was phosphorylated.
    • Show More Insertion of Ni promoter and ligation of 13-19-15
  • Ni inducible promoter was ligated into pCPC-3301 vector.
  • Phosphorylated 13 was ligated into pT-19-15 and transformed into E.coli via heat shock.
  • Single colonies were obtained by plating.
  • A colony PCR of Ni inducible promoter(pCPC-3301-Ni) was performed with 12 colonies.
    • Two of the successful ones were used to inoculate overnight cultures.
  • A colony PCR of pT-13-19-15 was performed with 7 colonies.
    • Two of the successful ones were used to inoculate overnight cultures.
  • Two kinds of plasmids were isolated using a miniprep kit.
    • Show More IMG_2956

    查看Team Tianjin全部实验 ›

    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

    查看Team Tianjin全部实验 ›

    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

    查看Team Tianjin全部实验 ›

    info_outline
    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