Team:Bielefeld-Germany/Results/Summary

From 2012.igem.org

(Difference between revisions)
Line 117: Line 117:
<h1>Datapage</h1>
<h1>Datapage</h1>
-
<p class="more">
 
</html>
</html>
__NOTOC__
__NOTOC__
Line 135: Line 134:
<html>
<html>
   
   
-
</p>
+
 
-
+
</div>
</div>
-
+
<div id="anzeige">
 +
 +
<h1>Laccases</h1>
 +
 +
 
 +
</html>
 +
Zusammenfassung
 +
 
 +
 
 +
<html>
 +
</div>
-
 
-
 
Line 155: Line 161:
<h1>Immobilization</h1>
<h1>Immobilization</h1>
-
<p class="more">
 
</html>
</html>
-
__NOTOC__
+
zusammenfassung
<html>
<html>
-
 
-
</p>
 
-
 
-
 
-
 
</div>
</div>
Line 175: Line 175:
<h1>Subtrate Analytics</h1>
<h1>Subtrate Analytics</h1>
-
               
 
-
 
-
<p class="more">
 
</html>
</html>
-
__NOTOC__
+
ZUsammenfassung
<html>
<html>
-
</p>
+
 
Line 192: Line 189:
<h1>Cellulose binding domain</h1>
<h1>Cellulose binding domain</h1>
-
<p class="more">
 
-
</p>
+
<html>
-
</html>
+
zusammenfassung
-
__NOTOC__
+
<html>
-
 
+
-
 
+
-
 
+
-
<html>
+
-
 
+
-
+
 +
</div>
</div>
<div id="anzeige">
<div id="anzeige">
<h1>Shuttle vector</h1>
<h1>Shuttle vector</h1>
-
 
+
</html>
-
<p class="more">
+
A shuttle vector for recombination into the yeast <i>P. pastoris</i> could be developed. With this system it is possible to recombine a protein of interest with a N-terminal mating factor alpha 1 for secretion the protein in the media. This protein of interest could be cloned in frame with one restiction-ligate-cloning-step. The selection depends not on an antibiotic resistance like zeocine, but on a complementation of histidine auxotrophy.  
A shuttle vector for recombination into the yeast <i>P. pastoris</i> could be developed. With this system it is possible to recombine a protein of interest with a N-terminal mating factor alpha 1 for secretion the protein in the media. This protein of interest could be cloned in frame with one restiction-ligate-cloning-step. The selection depends not on an antibiotic resistance like zeocine, but on a complementation of histidine auxotrophy.  
-
 
-
</html>
 
-
__NOTOC__
 
<html>
<html>
-
 
-
</p>
 
-
</html>
 
-
 
-
 
-
 
-
 
-
<html>
 
</div>
</div>
Line 233: Line 213:
<h1>Collaboration with UCL</h1>
<h1>Collaboration with UCL</h1>
-
<p class="more">
+
 
</html>
</html>
The BioBrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729002 BBa_K729002] from the [https://2012.igem.org/Team:University_College_London University&nbsp;College&nbsp;London] was characterized by us. Therefore ''E. coli'' KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729002 BBa_K729002] and ''E. coli'' KRX as negative control were cultivated in shaking flasks and a growth kinetic was determined. The harvested cells were lysed via [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Production#Sonification sonification] and the supernatant was purified from substances with a low molecular weight. After purification it was analyzed by [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#Sodium_dodecyl_sulfate_polyacrylamide_gel_electrophoresis_.28SDS-PAGE.29 SDS-PAGE], [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#Activity_measurements activity assay] as well as [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#MALDI MALDI-TOF].
The BioBrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729002 BBa_K729002] from the [https://2012.igem.org/Team:University_College_London University&nbsp;College&nbsp;London] was characterized by us. Therefore ''E. coli'' KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729002 BBa_K729002] and ''E. coli'' KRX as negative control were cultivated in shaking flasks and a growth kinetic was determined. The harvested cells were lysed via [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Production#Sonification sonification] and the supernatant was purified from substances with a low molecular weight. After purification it was analyzed by [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#Sodium_dodecyl_sulfate_polyacrylamide_gel_electrophoresis_.28SDS-PAGE.29 SDS-PAGE], [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#Activity_measurements activity assay] as well as [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#MALDI MALDI-TOF].
For a comparison ''E.&nbsp;coli'' KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K7863005] was cultivated and also analysed by [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#Sodium_dodecyl_sulfate_polyacrylamide_gel_electrophoresis_.28SDS-PAGE.29 SDS-PAGE] and [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#Activity_measurements activity assay]. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729002 BBa_K729002] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K7863005] showed a similar behaviour in oxidizing ABTS.
For a comparison ''E.&nbsp;coli'' KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K7863005] was cultivated and also analysed by [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#Sodium_dodecyl_sulfate_polyacrylamide_gel_electrophoresis_.28SDS-PAGE.29 SDS-PAGE] and [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Analytics#Activity_measurements activity assay]. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729002 BBa_K729002] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K7863005] showed a similar behaviour in oxidizing ABTS.
<html>
<html>
-
</html>
 
-
__NOTOC__
 
-
 
-
 
-
<html>
 
</div>
</div>
</div>
</div>

Revision as of 17:29, 26 September 2012

Results

Summary

Cultivation and Purification of the different laccases

During our research we cultivated the following BioBricks and produced several laccase. To simplify the presentation of our results we named the produced laccase like the following system

Produced and generated BioBricks with the source strain of the DNA-sequence, promoter, protein name and the names given by the iGEM Team Bielefeld
BioBrick code strain promoter name of protein name given by the iGEM Team
<partinfo>K863000</partinfo> Bacillus pumilus DSM 27 T7 promoter CotA BPUL
<partinfo>K863005</partinfo> E. coli BL21(DE3) T7 promoter CueO ECOL
<partinfo>K863010</partinfo> Thermus thermophilus HB27 T7 promoter tthL TTHL
<partinfo>K863012</partinfo> Thermus thermophilus constitutive promoter (<partinfo>BBa_J23100</partinfo>) tthL TTHL
<partinfo>K863015</partinfo> Xanthomonas campestris pv. campestris B100' T7 CopA XCCL
<partinfo>K863020</partinfo> Bacillus halodurans C-125 T7 Lbh1 BHAL
<partinfo>K863022</partinfo> Bacillus halodurans C-125 constitutive promoter (<partinfo>BBa_J23100</partinfo>) Lbh1 BHAL


All BioBricks of the iGEM Team Bielefeld were screened to identify the best conditions for protein expression. The first trials were made by shaking flask cultivations with different parameters. These parameters were various shaking flask designs , different temperatures, different concentrations of chloramphenicol, various induction strategies , several cultivation times and some cultivations in absence or presence of CuCl2. To detect the produced laccases different analysis methods were performed like SDS-PAGE analysis as well as MALDI-TOF.





Datapage


How our system works

Data for our favorite new parts

  1. <partinfo>K863000</partinfo> - bpul (laccase from Bacillus pumilus) with T7 promoter, RBS and HIS tag:
  2. <partinfo>K863005</partinfo> - ecol (laccase from E. coli) with T7 promoter, RBS and HIS tag:

Data for pre-existing parts

We have also characterized the following parts

  1. <partinfo>BBa_K863012</partinfo> - tthl laccase (from T. thermophilus) with constitutive promoter J23100, RBS and HIS tag:
  2. <partinfo>BBa_K863022</partinfo> - bhal laccase (from Bacillus halodurans) with constitutive promoter J23100, RBS and HIS tag:

Laccases

Zusammenfassung


Immobilization

zusammenfassung


Subtrate Analytics

ZUsammenfassung


Cellulose binding domain

zusammenfassung

Shuttle vector

A shuttle vector for recombination into the yeast P. pastoris could be developed. With this system it is possible to recombine a protein of interest with a N-terminal mating factor alpha 1 for secretion the protein in the media. This protein of interest could be cloned in frame with one restiction-ligate-cloning-step. The selection depends not on an antibiotic resistance like zeocine, but on a complementation of histidine auxotrophy.

Collaboration with UCL

The BioBrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729002 BBa_K729002] from the University College London was characterized by us. Therefore E. coli KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729002 BBa_K729002] and E. coli KRX as negative control were cultivated in shaking flasks and a growth kinetic was determined. The harvested cells were lysed via sonification and the supernatant was purified from substances with a low molecular weight. After purification it was analyzed by SDS-PAGE, activity assay as well as MALDI-TOF. For a comparison E. coli KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K7863005] was cultivated and also analysed by SDS-PAGE and activity assay. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729002 BBa_K729002] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K7863005] showed a similar behaviour in oxidizing ABTS.


55px Logo merck.jpg BioCircle.JPG Bielefeld2012 Evonik.jpg Bielefeld2012 Baxter.png Logo knauer.jpg Logo iit.jpg Bielefeld2012 BIEKUBA.jpg Logo biometra.jpg Logo bio-nrw.png Bielefeld2012 Logo ERASynbio.jpg