Team:Bielefeld-Germany/Test

From 2012.igem.org

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{{Team:Bielefeld/Head}}
{{Team:Bielefeld/Head}}
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=Feedback: Analysis of the Feedback from Street Science=
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==>Week 24 (10/08 - 10/14/12)</h1>==
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<html>
 
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<link rel="stylesheet" href="http://2012.igem-bielefeld.de/includes/wiki/flex/css/flexslider.css" type="text/css">
 
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<script src="https://ajax.googleapis.com/ajax/libs/jquery/1.6.2/jquery.min.js"></script>
 
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<script src="http://2012.igem-bielefeld.de/includes/wiki/flex/js/jquery.flexslider.js"></script>
 
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<!-- Load the flexslider -->
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===Monday October 08th===
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<script type="text/javascript" charset="utf-8">
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All:
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  $(window).load(function() {
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Day off in Amsterdam!!!
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    $('.flexslider').flexslider({
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          animation: "fade",  // slide or fade
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          slideshow: false,
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<!-- Slider Container and Images -->
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===Tuesday October 09th===
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<div style="padding:5px; z-index:1;">
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===Wednesday October 10th===
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* '''Team Shuttle Vector:'''
 +
** Ligation of pSB1C3::BBa_K863202 construct in KRX cells was done.
 +
* '''Team Cellulose Binding Domain:'''
 +
** Gradient-PCRs of CBDcex_Freiburg; CBDclos_Freiburg; GFP_Freiburg and ecol_Freiburg (47 °C to 67°C)
 +
*** Product:
 +
**** CBDcex_Freiburg at all temperatures
 +
**** CBDclos_Freiburg at all temperatures
 +
**** GFP_Freiburg at all temperatures
 +
**** ecol_Freiburg at 53 °C to 61°C (best at 57 to 59 °C)
 +
*** pooled fractions for gel clean up
 +
* '''Team Site Directed Mutagenesis:'''
 +
** Ordered Primers for the SDM of the illegal XbaI restriction site in the shuttle vector
 +
* '''Team Cultivation and Purification:'''
 +
**Made precultures of ''E.&nbsp;coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005]for 19L fermentation (500mL preculture)
 +
===Thursday October 11th===
 +
* '''Team Cellulose Binding Domain:'''
 +
** Clean-Up of the Gel-pieces (from PCR-products)
 +
** Restriction of PCR-products (3h):
 +
*** GFP_Freiburg:
 +
**** XbaI+PstI Tango-Buffer for Ligation
 +
**** XbaI+AgeI Tango 4xAgeI for Assembly
 +
*** CBDclos/cex:
 +
**** NgoMIV+PstI P.4+BSA 2xPstI for Assembly
 +
** Assembly and Ligation (1h with T4):
 +
*** GFP_Freiburg + CBDcex_Freiburg + J61101
 +
*** GFP_Freiburg + CBDclos_Freiburg + J61101
 +
*** GFP_Freiburg + J61101
 +
** Transformation in ''E. coli'' KRX (3 µL, chemical):
 +
*** GFP_Freiburg + CBDcex_Freiburg + J61101
 +
*** GFP_Freiburg + CBDclos_Freiburg + J61101
 +
*** GFP_Freiburg + J61101
 +
** All Transformations were plated on AMP-select-agar
 +
*'''Team Cultivation and Purification'''
 +
** 12 L Cultivation of ''E.&nbsp;coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005]
 +
***Settings:
 +
Bioengineering NFL 19L fermenter, autoinduction HSG medium, 60 µg/mL chloramphenicol, 19 L, 37 °C, stirrer on cascade to hold a pO2 of 50 %, airflow: 12 NL/m, 16 hours. HSG medium was choosen to get a high biomass concentration with hope for a higher amount of laccases.
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        <div class="flexslider" style="margin-top:10px;">
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**Made precultures of ''E.&nbsp;coli'' Rosetta Gami 2 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012](09/09) and <partinfo>BBa_K863022</partinfo> (09/09) behind a constitutive promoter. (500mL preculture)
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            <ul class="slides">
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                  <li>
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                    <div class="frontpage-slide">
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                        <a href=""><img src="https://static.igem.org/mediawiki/2012/3/3a/Bielefeld2012_Occupation.jpg"></a>
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                        <div class="slide-right-col">
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                            Occupation of the participants. 25% of the participants were pupils and 29% were students and 21% were workers. Altogether, the participants had an positive impression on synthetic biology and in all cases participants were judging the happening as very well run and would visit a „Street-Science“-event again.
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===Friday October 12th===
 +
* '''Team Cellulose Binding Domain:'''
 +
** A lot of colonies on all three dishes (J61101+GFP_Freibug;J61101+GFP_Freibug+CBDcex;J61101+GFP_Freibug+CBDclos)
 +
*** no obvious green fluorescence, not even at J61101+GFP
 +
*** Maybe the combination of J23100 and J61101 isn't strong enough to express a visible amount of GFP
 +
** Colony-PCR of 16 colonies of every dish
 +
*** J61101 + GFP_Freiburg:
 +
**** 16/16 positive
 +
**** plated two on select-agar
 +
*** J61101 + GFP_Freiburg + CBDcex:
 +
**** 8/16 positve
 +
**** plated three on select-agar
 +
*** J61101 + GFP_Freiburg + CBDclos:
 +
**** 7/16 positive
 +
**** plated three on select-agar
 +
* '''Team Shuttle Vector:'''
 +
** The induction with 0.5% (v/v) methanol of P. pastoris GS115 cells with integrated BBa_K863204::GFP was started.
 +
* '''Team Substrate Analysis''':
 +
** Since we have seen some new peaks after Estradiol treated with Laccases in the LC-MS, we wanted to have more degradation products to do an MS so we used a higher concentradion of Estradiol and Laccase and let them incubate for 66 hours.
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                        </div>
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* '''Team Cultivation and Purification'''
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                        <div class=clear></div>
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** 12L Cutlivations of ''E.&nbsp;coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005] were harvested and stored at 4°C until purification.  
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                    </div>
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** 12 L Cultivation of ''E.&nbsp;coli'' Rosetta Gami 2 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012](09/09) and <partinfo>BBa_K863022</partinfo> (09/09)
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                </li>
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*** Settings:
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                <li>
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Bioengineering NFL 19L fermenter, HSG medium, 60 µg/mL chloramphenicol, 300 µg/mL ampicillin, 12 L, 37 °C, stirrer on cascade to hold a pO2 of 50 %, airflow: 12 NL/m, 22-24 hours. HSG medium was chosen to get a high biomass concentration with hope for a higher amount of laccases.
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                    <div class="frontpage-slide">
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                        <a href=''><img src="https://static.igem.org/mediawiki/2012/9/92/Bielefeld2012_Howdidyoufind.jpg"></a>
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                        <div class="slide-right-col">
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                            <p>
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                              58,33 of the participants found "Street-Science" regarding to synthetic Biology interesting and 41,67% found it informative, 20,83% found Street-Science exeptionally and 33,33% found it innovative. Moreover 29,17% of the participants found it cosmopolitan and 41,67% found this event helpful.
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                            </p>
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                        </div>
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                        <div class=clear></div>
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                    </div>
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                </li>
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                <li>
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                    <div class="frontpage-slide">
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                        <a href=""><img src="https://static.igem.org/mediawiki/2012/6/64/Bielefeld2012_Questions.jpg"></a>
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                        <div class="slide-right-col">
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                          48% indicated, that everything could be answered, 38% of the participants indicated that almost could be answered, and 14% indicated, that the most of the question could be answered.
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                        </div>
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===Saturday October 13th===
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                        <div class=clear></div>
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* '''Team Shuttle Vector:'''
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                    </div>
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** The P. pastoris GS115 cells integrated with BBa_K863204::GFP were also inducted with 0.5% (v/v) methanol (100%).
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                </li>
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                <li>
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                    <div class="frontpage-slide">
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                        <a href=""><img src="https://static.igem.org/mediawiki/2012/0/03/Relation.jpg"></a>
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                        <div class="slide-right-col">
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                            The participants can choose from the scale 1(absolutely not true) till 7 (absolutely true) to evaluate the following statements. The values are average values.
+
 +
* '''Team Cultivation and Purification'''
 +
** 12L Cutlivations of ''E.&nbsp;coli'' Rosetta Gami 2 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012](09/09) and <partinfo>BBa_K863022</partinfo> (09/09) were harvested and stored at 4°C until purification.
-
                        </div>
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===Sunday October 14th===
 +
* '''Team Shuttle Vector''':
 +
** The P. pastoris GS115 cells integrated with BBa_K863204::GFP were also inducted with 0.5% (v/v) methanol (100%).
-
                        <div class=clear></div>
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* '''Team Fungal and Plant Laccases:'''
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                    </div>
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** Plasmid isolation of pSB1C3::BBa_K863204::TV5 for transformation into P. pastoris GS115 was done. After isolation the plasmid was digested with [[Team:Bielefeld-Germany/Protocols/Materials#Used_enzymes |NotI]], purified with the PCR clean up kit and transformed via [[Team:Bielefeld-Germany/Protocols/molecular_genetics#Transformation_of_yeast_cells |protocol]] in P. pastoris GS115.
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                </li>
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                <li>
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                    <div class="frontpage-slide">
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                        <a href=""><img src="https://static.igem.org/mediawiki/2012/9/98/Bielefeld2012_Opinion1.jpg"></a>
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                        <div class="slide-right-col">
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The participants can choose from scale 1 (absolutely not true) till 7 (totally true). 4,21 is the average value of the participants, who are positively suprised about the project and about synthetic biology.
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* '''Team Cellulose Binding Domain:'''
-
                        <div class=clear></div>
+
** Tried to isolate all plates, but only got low concentrations (5 ng/µL - 20 ng/µL)
-
                    </div>
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** plated eight different colonies for isolation
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                </li>
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            </ul>
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        </div>
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    </div>
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* '''Team Substrate Analysis''':
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</html>
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** We took sample from the reaction of the 12th October.
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==Evaluation==
 
-
<div style="text-align:justify;">
 
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On the event, called „Street-Science“, the iGEM-Team 2012 of Bielefeld University invited people to isolate DNA from vegetable and fruits. Furthermore games like „Spitzen stecken“ (sticking pipette-tips) were provided for kids and who ever wanted to have a try or challenge one of our team. The concern of our team was to talk about synthetic biology, show its opportunities and help dismantle prejudice against it.
 
-
To get a Feedback about the event, we planned an evaluation survey, which the participants could fill out anonymously. The results we got from their suggestions, ideas and critics, should help us reflect unbiased about the work we had done. Moreover, based on the questions of the survey, it was to find out whether we had successfully submitted our project and the means of synthetic biology to the people. In addition we wanted to know what impression our team left on the participants.
 
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Overall, the surveys were filled out by 24 participants, divided in 33% of women and 66% of men. Most of the participants were students, pupils and workers.
 
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Several proposals were given, that we should plan more room for the event and do even more experiments at a time. Also the people ask for a donation box. In general the people considered that they had met a group of friendly students, which are able to discuss about problems and questions of synthetic biology. Furthermore it was proposed to make an event like „Street-Science“ at schools.
 
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The original Feedback-Interview can you find [https://static.igem.org/mediawiki/2012/e/e4/Bielefeld2012_Preview.pdf here].
 
{{Team:Bielefeld/Sponsoren}}
{{Team:Bielefeld/Sponsoren}}

Revision as of 09:52, 26 October 2012

Contents

>Week 24 (10/08 - 10/14/12)</h1>

Monday October 08th

All: Day off in Amsterdam!!!

Tuesday October 09th

Wednesday October 10th

  • Team Shuttle Vector:
    • Ligation of pSB1C3::BBa_K863202 construct in KRX cells was done.
  • Team Cellulose Binding Domain:
    • Gradient-PCRs of CBDcex_Freiburg; CBDclos_Freiburg; GFP_Freiburg and ecol_Freiburg (47 °C to 67°C)
      • Product:
        • CBDcex_Freiburg at all temperatures
        • CBDclos_Freiburg at all temperatures
        • GFP_Freiburg at all temperatures
        • ecol_Freiburg at 53 °C to 61°C (best at 57 to 59 °C)
      • pooled fractions for gel clean up
  • Team Site Directed Mutagenesis:
    • Ordered Primers for the SDM of the illegal XbaI restriction site in the shuttle vector
  • Team Cultivation and Purification:

Thursday October 11th

  • Team Cellulose Binding Domain:
    • Clean-Up of the Gel-pieces (from PCR-products)
    • Restriction of PCR-products (3h):
      • GFP_Freiburg:
        • XbaI+PstI Tango-Buffer for Ligation
        • XbaI+AgeI Tango 4xAgeI for Assembly
      • CBDclos/cex:
        • NgoMIV+PstI P.4+BSA 2xPstI for Assembly
    • Assembly and Ligation (1h with T4):
      • GFP_Freiburg + CBDcex_Freiburg + J61101
      • GFP_Freiburg + CBDclos_Freiburg + J61101
      • GFP_Freiburg + J61101
    • Transformation in E. coli KRX (3 µL, chemical):
      • GFP_Freiburg + CBDcex_Freiburg + J61101
      • GFP_Freiburg + CBDclos_Freiburg + J61101
      • GFP_Freiburg + J61101
    • All Transformations were plated on AMP-select-agar
  • Team Cultivation and Purification

Bioengineering NFL 19L fermenter, autoinduction HSG medium, 60 µg/mL chloramphenicol, 19 L, 37 °C, stirrer on cascade to hold a pO2 of 50 %, airflow: 12 NL/m, 16 hours. HSG medium was choosen to get a high biomass concentration with hope for a higher amount of laccases.

    • Made precultures of E. coli Rosetta Gami 2 containing BBa_K863012(09/09) and <partinfo>BBa_K863022</partinfo> (09/09) behind a constitutive promoter. (500mL preculture)

Friday October 12th

  • Team Cellulose Binding Domain:
    • A lot of colonies on all three dishes (J61101+GFP_Freibug;J61101+GFP_Freibug+CBDcex;J61101+GFP_Freibug+CBDclos)
      • no obvious green fluorescence, not even at J61101+GFP
      • Maybe the combination of J23100 and J61101 isn't strong enough to express a visible amount of GFP
    • Colony-PCR of 16 colonies of every dish
      • J61101 + GFP_Freiburg:
        • 16/16 positive
        • plated two on select-agar
      • J61101 + GFP_Freiburg + CBDcex:
        • 8/16 positve
        • plated three on select-agar
      • J61101 + GFP_Freiburg + CBDclos:
        • 7/16 positive
        • plated three on select-agar
  • Team Shuttle Vector:
    • The induction with 0.5% (v/v) methanol of P. pastoris GS115 cells with integrated BBa_K863204::GFP was started.
  • Team Substrate Analysis:
    • Since we have seen some new peaks after Estradiol treated with Laccases in the LC-MS, we wanted to have more degradation products to do an MS so we used a higher concentradion of Estradiol and Laccase and let them incubate for 66 hours.
  • Team Cultivation and Purification
    • 12L Cutlivations of E. coli KRX with BBa_K863000 and BBa_K863005 were harvested and stored at 4°C until purification.
    • 12 L Cultivation of E. coli Rosetta Gami 2 containing BBa_K863012(09/09) and <partinfo>BBa_K863022</partinfo> (09/09)
      • Settings:

Bioengineering NFL 19L fermenter, HSG medium, 60 µg/mL chloramphenicol, 300 µg/mL ampicillin, 12 L, 37 °C, stirrer on cascade to hold a pO2 of 50 %, airflow: 12 NL/m, 22-24 hours. HSG medium was chosen to get a high biomass concentration with hope for a higher amount of laccases.

Saturday October 13th

  • Team Shuttle Vector:
    • The P. pastoris GS115 cells integrated with BBa_K863204::GFP were also inducted with 0.5% (v/v) methanol (100%).
  • Team Cultivation and Purification
    • 12L Cutlivations of E. coli Rosetta Gami 2 containing BBa_K863012(09/09) and <partinfo>BBa_K863022</partinfo> (09/09) were harvested and stored at 4°C until purification.

Sunday October 14th

  • Team Shuttle Vector:
    • The P. pastoris GS115 cells integrated with BBa_K863204::GFP were also inducted with 0.5% (v/v) methanol (100%).
  • Team Fungal and Plant Laccases:
    • Plasmid isolation of pSB1C3::BBa_K863204::TV5 for transformation into P. pastoris GS115 was done. After isolation the plasmid was digested with NotI, purified with the PCR clean up kit and transformed via protocol in P. pastoris GS115.
  • Team Cellulose Binding Domain:
    • Tried to isolate all plates, but only got low concentrations (5 ng/µL - 20 ng/µL)
    • plated eight different colonies for isolation
  • Team Substrate Analysis:
    • We took sample from the reaction of the 12th October.




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About our Wiki

Fifteen students from different fields of study from the Bielefeld University aspired to complete a project called TOXIC COMPOUNDS IN NATURAL WATER - A CASE FOR LACCASE. In his Wiki we document our achievements and our approach.

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