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

 
(5 intermediate revisions by one other user not shown)
Line 5: Line 5:
  
 
<html lang="en">
 
<html lang="en">
 +
<style>
 +
#Notes{
 +
  color: inherit;
 +
  background-color: rgba(255, 255, 255, 0.1);
 +
}
 +
</style>
 +
 
<head>
 
<head>
 
<meta charset="utf-8" />  
 
<meta charset="utf-8" />  
Line 219: Line 226:
  
 
   
 
   
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/9/97/T--Tianjin--cf-blank.png"  alt="T--Tianjin--cf-blank" width="200px">
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/9/97/T--Tianjin--cf-blank.png"  alt="T--Tianjin--cf-blank" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/d/d0/T--Tianjin--cf-m1.png"  alt="T--Tianjin--cf-m1" width="200px">
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/d/d0/T--Tianjin--cf-m1.png"  alt="T--Tianjin--cf-m1" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m2.png"  alt="T--Tianjin--cf-m2" width="200px">
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/4/44/T--Tianjin--cf-m2.png"  alt="T--Tianjin--cf-m2" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/34/T--Tianjin--cf-m3.png"  alt="T--Tianjin--cf-m3" width="200px">
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/34/T--Tianjin--cf-m3.png"  alt="T--Tianjin--cf-m3" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/7/75/T--Tianjin--cf-m4.png"  alt="T--Tianjin--cf-m4" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/7/75/T--Tianjin--cf-m4.png"  alt="T--Tianjin--cf-m4" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/b/be/T--Tianjin--cf-m5.png"  alt="T--Tianjin--cf-m5" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/b/be/T--Tianjin--cf-m5.png"  alt="T--Tianjin--cf-m5" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/4/4e/T--Tianjin--cf-m6.png"  alt="T--Tianjin--cf-m6" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/4/4e/T--Tianjin--cf-m6.png"  alt="T--Tianjin--cf-m6" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/4/4e/T--Tianjin--cf-m7.png"  alt="T--Tianjin--cf-m7" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/4/4e/T--Tianjin--cf-m7.png"  alt="T--Tianjin--cf-m7" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/9/97/T--Tianjin--cf-m8.png"  alt="T--Tianjin--cf-m8" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/9/97/T--Tianjin--cf-m8.png"  alt="T--Tianjin--cf-m8" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/d/d1/T--Tianjin--cf-m9.png"  alt="T--Tianjin--cf-m9" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/d/d1/T--Tianjin--cf-m9.png"  alt="T--Tianjin--cf-m9" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/6/61/T--Tianjin--cf-m10.png"  alt="T--Tianjin--cf-m10" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/6/61/T--Tianjin--cf-m10.png"  alt="T--Tianjin--cf-m10" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/c/c9/T--Tianjin--cf-m11.png"  alt="T--Tianjin--cf-m11" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/c/c9/T--Tianjin--cf-m11.png"  alt="T--Tianjin--cf-m11" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/36/T--Tianjin--cf-m12.png"  alt="T--Tianjin--cf-m12">
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/36/T--Tianjin--cf-m12.png"  alt="T--Tianjin--cf-m12"height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/d/d2/T--Tianjin--cf-m13.png"  alt="T--Tianjin--cf-m13" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/d/d2/T--Tianjin--cf-m13.png"  alt="T--Tianjin--cf-m13" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/35/T--Tianjin--cf-m14.png"  alt="T--Tianjin--cf-m14" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/35/T--Tianjin--cf-m14.png"  alt="T--Tianjin--cf-m14" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/b/b3/T--Tianjin--cf-m15.png"  alt="T--Tianjin--cf-m15" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/b/b3/T--Tianjin--cf-m15.png"  alt="T--Tianjin--cf-m15" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/2/2c/T--Tianjin--cf-m16.png"  alt="T--Tianjin--cf-m16" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/2/2c/T--Tianjin--cf-m16.png"  alt="T--Tianjin--cf-m16" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/9/91/T--Tianjin--cf-m17.png"  alt="T--Tianjin--cf-m17" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/9/91/T--Tianjin--cf-m17.png"  alt="T--Tianjin--cf-m17" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/3f/T--Tianjin--cf-m18.png"  alt="T--Tianjin--cf-m18" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/3f/T--Tianjin--cf-m18.png"  alt="T--Tianjin--cf-m18" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/a/ad/T--Tianjin--cf-m19.png"  alt="T--Tianjin--cf-m19" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/a/ad/T--Tianjin--cf-m19.png"  alt="T--Tianjin--cf-m19" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/0/03/T--Tianjin--cf-m20.png"  alt="T--Tianjin--cf-m20" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/0/03/T--Tianjin--cf-m20.png"  alt="T--Tianjin--cf-m20" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/b/b9/T--Tianjin--cf-m21.png"  alt="T--Tianjin--cf-m21" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/b/b9/T--Tianjin--cf-m21.png"  alt="T--Tianjin--cf-m21" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/3d/T--Tianjin--cf-m22.png"  alt="T--Tianjin--cf-m22" >
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/3d/T--Tianjin--cf-m22.png"  alt="T--Tianjin--cf-m22" height="250px">
−
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/3e/T--Tianjin--cf-m-no.png"  alt="T--Tianjin--cf-m-no">
+
<img class="col-md-4" src="https://static.igem.org/mediawiki/2016/3/3e/T--Tianjin--cf-m-no.png"  alt="T--Tianjin--cf-m-no"height="250px">
 +
 
 
<br/>
 
<br/>
  
−
+
<p>
 
<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>   
 
<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>   
 +
</p>
  
 
          
 
          

Latest revision as of 14:07, 19 October 2016

TEAM TIANJIN

LOGO


Team Tianjin-Attribution

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.

    • Show More

    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.

    • Show More

    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

    • Show More

    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.

    Show More

    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.

    • Show More

    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.
    Show More
    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

    Team Tianjin Sponsor Alltech
    Team Tianjin Sponsor GenScript
    Team Tianjin Sponsor SynbioTech