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

Line 216: Line 216:
  
 
<li><b>Parallel experiments for expression in CFPS system<b></li><br/>
 
<li><b>Parallel experiments for expression in CFPS system<b></li><br/>
 +
 +
<div class="note-content">
 
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/9/97/T--Tianjin--cf-blank.png"  alt="T--Tianjin--cf-blank" width="800" height="533">
 
<img class="aligncenter size-full wp-image-4256" src="https://static.igem.org/mediawiki/2016/9/97/T--Tianjin--cf-blank.png"  alt="T--Tianjin--cf-blank" 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/d/d0/T--Tianjin--cf-m1.png"  alt="T--Tianjin--cf-m1" width="800" height="533">
Line 239: Line 241:
 
<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/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/2016/3/3d/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/2016/3/3d/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/2016/3/3e/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/2016/3/3e/T--Tianjin--cf-m-no.png"  alt="T--Tianjin--cf-m-no" width="800" height="533"><br/>
  
 +
</div>
 +
<a class="expand-btn">Show More</a>
  
 +
<b>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.<b>
  
−
<div class="note-content3">
 
  
  
  
−
<li><i>13</i> amplification at 65.0°C with 13.rev/fwd primes</li>
 
−
PCR worked, positive control worked, no amplification of <i>13</i></li>
 
−
<li>The fragments of <i>13</i> were purified with PCR Purification Kit.</li>
 
−
<li><i>13</i> gene fragment was phosphorylated.</li>
 
−
         
 
−
       
 
−
 
−
</div>
 
−
<a class="expand-btn3">Show More</a>
 
−
 
−
 
−
<b>Insertion of Ni promoter and ligation of <i>13-19-15</i></b><br/>
 
−
 
−
<div class="note-content4">
 
−
 
−
 
−
<li>Ni inducible promoter was ligated into pCPC-3301 vector.</li>
 
−
<li>Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.</li>
 
−
<li>Single colonies were obtained by plating.</li>
 
−
<li>A colony PCR of Ni inducible promoter(pCPC-3301-Ni) was performed with 12 colonies.</li>
 
−
Two of the successful ones were used to inoculate overnight cultures.</li>
 
−
<li>A colony PCR of pT-13-19-15 was performed with 7 colonies.</li>
 
−
  Two of the successful ones were used to inoculate overnight cultures.</li>
 
−
<li>Two kinds of plasmids were isolated using a miniprep kit.</li>
 
−
 
−
 
−
         
 
−
       
 
−
</div>
 
−
            <a class="expand-btn4">Show More</a>
 
−
 
−
 
−
<a href="https://www.camarts.cn/archives/4252.html"><img class="aligncenter size-full wp-image-4256" src="http://static.camarts.cn/images/spinner-1600.gif"  alt="IMG_2956" width="800" height="533" srcset="images/img_1.jpg 800w, http://img.camarts.cn/2016/01/IMG_2956-150x100.jpg 150w, http://img.camarts.cn/2016/01/IMG_2956-300x200.jpg 300w, http://img.camarts.cn/2016/01/IMG_2956-768x512.jpg 768w, http://img.camarts.cn/2016/01/IMG_2956-450x300.jpg 450w" sizes="(max-width: 800px) 100vw, 800px" /><noscript><img class="aligncenter size-full wp-image-4256" src="http://img.camarts.cn/2016/01/IMG_2956.jpg?imageView/2/w/800/h/800/q/90" alt="IMG_2956" width="800" height="533" srcset="http://img.camarts.cn/2016/01/IMG_2956.jpg 800w, http://img.camarts.cn/2016/01/IMG_2956-150x100.jpg 150w, http://img.camarts.cn/2016/01/IMG_2956-300x200.jpg 300w, http://img.camarts.cn/2016/01/IMG_2956-768x512.jpg 768w, http://img.camarts.cn/2016/01/IMG_2956-450x300.jpg 450w" sizes="(max-width: 800px) 100vw, 800px" /></noscript></a></p>
 
 
<p> <a href="https://www.camarts.cn/archives/4252.html" class="more-link">查看Team Tianjin全部实验 <span class="meta-nav">&rsaquo;</span></a></p>
 
<p> <a href="https://www.camarts.cn/archives/4252.html" class="more-link">查看Team Tianjin全部实验 <span class="meta-nav">&rsaquo;</span></a></p>
 
</div><!-- .entry-content -->
 
</div><!-- .entry-content -->

Revision as of 05:53, 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
    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.

    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