Team:Bielefeld-Germany/Labjournal/week20

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{{Team:Bielefeld/Head}}
{{Team:Bielefeld/Head}}
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==Week 20 (09/10 - 09/16/12)==
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<style type="text/css">
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===Monday September 10th===
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ul {list-style-image:none;}
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* '''Team Site Directed Mutagenesis:'''
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#bodyContent{
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**Digestion of the six tvel10-plasmids. Three were unmutated and other three hadn't lost the illegal ''Spe''I-restriction-site, but their second fragment was of a smaller size.
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    background-color: white;
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**plated three additional tvel-t243g-colonies.
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}
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* '''Team Cellulose Binding Domain:'''
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</style>
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**Sequencing results arrived: One [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)] is completely right!
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** Nanodropping plasmids of isolated [http://partsregistry.org/Part:BBa_K863113 CBDclos(T7)+GFP_His] showed that the cells did not have a plasmid at all (selection-agar did not work)
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** Gradient-PCR with [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)] as template and [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg]-primers did work just fine (best temperature 61,7°C); Digestion with ''Xba''I and ''Age''I.
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**Gel-Clean-up of [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg] and [http://partsregistry.org/Part:BBa_K863121 GFP_His]
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** Digestion of [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg] with ''Xba''I+''Age''I.
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** Digestion of [http://partsregistry.org/Part:BBa_K863121 GFP_His] with ''Ngo''MIV and ''Pst''I.
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* '''Team Cultivation & Purification:'''
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<!-- navigator -->
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** Cell disruption via high-pressure homogeniser and purification via Ni-NTA column were performed for the following samples: 3L fermentation of ''E.coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] (09/09), 6L fermentation of ''E.coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005] (09/07) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] (09/09).
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<div id="nav" class="tabs">
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** We made SDS-Pages of purification of ECOL.
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<div class="scroller">
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<ul style="list-style-type:none">
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/Starting"><strong>Prologue</strong></a></li>
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                <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week1"><strong>Week 1</strong></a></li>
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                <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week2"><strong>Week 2</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week3"><strong>Week 3</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week4"><strong>Week 4</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week5"><strong>Week 5</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week6"><strong>Week 6</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week7"><strong>Week 7</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week8"><strong>Week 8</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week9"><strong>Week 9</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week10"><strong>Week 10</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week11"><strong>Week 11</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week12"><strong>Week 12</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week13"><strong>Week 13</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week14"><strong>Week 14</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week15"><strong>Week 15</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week16"><strong>Week 16</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week17"><strong>Week 17</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week18"><strong>Week 18</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week19"><strong>Week 19</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week20"><strong>Week 20</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week21"><strong>Week 21</strong></a></li>
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<li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week22"><strong>Week 22</strong></a></li>
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===Tuesday September 11th===
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</ul>
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* '''Fungal Laccases:'''
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**PCR on tvel5 laccase for cloning in shuttle vector. Digest of shuttle vector and digest of tvel35 with ''Aar''I enzyme.
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* '''Team Shuttle Vector:'''
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</div>
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**Digest of shuttle shuttle vector with ''Pvu''II and ''Hind''III as control. Agarose gel looks good.
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</div>
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</html>
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* '''Team Cellulose Binding Domain:'''
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<div style="text-align:justify;">
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** Assembly of <partinfo>BBa_K863104</partinfo>  and <partinfo>BBa_K863114</partinfo>:
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*** Assembled [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg] and [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg] with [http://partsregistry.org/Part:BBa_K863121 GFP_His] and <partinfo>J61101</partinfo> and plated the ligation on AMP-selection-agar (because of the pSB1A2-backbone).
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** Restriktion of [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg] and [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg] with ''Eco''RI and ''Pst''I.
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*** Ligation of CBDcex_Freiburg  and CBDclos_Freiburg with the pSB1C3-backbone and transformated and plated on selection-agar
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* '''Team Cultivation & Purification:'''
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==Week 20 (09/10 - 09/16/12)==
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** Made precultures of ''E.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] as well as of ''E.coli'' KRX.
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===Wednesday September 12th===
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* '''Team Site Directed Mutagenesis:'''
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**Plasmid-isolation of the three tvel10-plasmids and digestion with ''Spe''I showed two unmutated plasmids and one with the same wrong restriction-fragments as Monday. There must be a systematical error. ''pfu''-PCR should be done again.
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* '''Team Cellulose Binding Domain:'''
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** There are only few colonies on all selection-agar-dishes, but none is obviously green fluoresensing, even with UV-light emission could not be stimulated.
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** Plated colonies of [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg] and [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg] to see if they are red or not.
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** Colony-PCR of [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg] and [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg] showed eight positive colonies to the <partinfo>K863104</partinfo>-insert. Plated the positive colonies for plasmid-isolation.
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*'''Team Cultivation & Purification'''
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** Cell disruption via sonification and purification via Ni-NTA column were performed for the following samples: 200 mL cultivation of ''E.coli'' Rosetta Gami 2 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012](09/09) and laccase from ''B.halodurans'' (09/09) behind a constitutive promotor.
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** SDS-Pages of the flask cultivation from 09/09 ( ''E.coli'' Rosetta Gami 2 containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012](09/09) and laccase from ''B.halodurans'')
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===Thursday September 13th===
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* '''Team Cellulose Binding Domain:'''
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** Designed a lot of Primers to cope with the expression problem. E.g. inserting a long S2N10 Linker between the CBD and the GFP, also getting rid of the His-tag on the GFP to easily change the order of CBD and GFP.
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** Plasmid-isolation of <partinfo>BBa_K863104</partinfo>-transformation clones
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** Colony-PCR of [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg] and [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg] colonies. All [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg]-colonies are positive and half of the [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg]-colonies.
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** Colony-PCR of CBDclos_F.+GFP with no positive result
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** Colony-PCR of [http://partsregistry.org/Part:BBa_K863122 const.GFP_His]: 2 positive (one fluoreszend); Plated both positive and one additional fluorescend.
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* '''Team Cultivation & Purification:'''
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** Fermentation of ''E. coli'' KRX withoud plasmid (fermenter: Infors) and with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] (fermenter: Braun Biostat)
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*** Settings: fermenter: Infors/Braun Biostat, final volume: 3 L, autoinduction medium, 60 µg/mL chloramphenicol added, 37 °C, stirrer on cascade to hold a pO2 of 50 %, airflow: 2 NL/m, durance: 12 h.
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** Made preculture of ''E. coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863103 BBa_K863103] (CBD-GFP-His).
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** Made preculture of ''P. pastoris'' GS115
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** Fermentation of ''E. coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000].
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*** Settings: fermenter: Bioengineering NFL22(7 L), final volume: 6 L, autoinduction medium with 60 µg/mL chloramphenicol added, 37 °C, stirrer increased 2 % if the pO2 got below 30 %, airflow: 5 NF/m, 12 hours.
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===Friday September 14th===
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* '''Team Cellulose Binding Domain:'''
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** Isolated three glowing dishes of KRX with the [http://partsregistry.org/Part:BBa_K863122 const.GFP_His]-plasmid, three with the [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg]-plasmid and [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg]-plasmid.
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* '''Team Cultivation & Purification: '''
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** Cell disruption of fermentation 09/13 via high-pressure homogenizer and purification via Ni-NTA column. Made SDS-Pages of purificated fractions.
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** Repeat the preculture of ''P. pastoris'' GS115, because of using the wrong media.
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** Made preculure of ''E. coli'' Rosetta Gami 2 with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012].
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===Saturday September 15th===
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* '''Team Cellulose Binding Domain:'''
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** KRX culture of [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)-GFP_His] seems to have a green glow.
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*** Isolation of four pellets of [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)-GFP_His] for us and SDU Denmark.
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** [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)] digested with ''Spe''I and ''Age''I and deposphorylated.
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** [http://partsregistry.org/Part:BBa_K863121 GFP_His]-PCR-product (gel-clean) digested with ''Spe''I and ''Ngo''MIV.
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** Ligated [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)] with [http://partsregistry.org/Part:BBa_K863121 GFP_His] and plated on select-Agar.
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** Restriction-analysis showed that all [http://partsregistry.org/Part:BBa_K863122 const.GFP_His]-plasmids are correct, as are the three [http://partsregistry.org/Part:BBa_K863111 CBDclos] and two of the [http://partsregistry.org/Part:BBa_K863101 CBDcex]
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** Collected data to make a protocol for a Cellulose binding assay:
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*** Avicel: about 11,4 mg protein (CBD) binds to 1 g Avicel (0,14 mg/12,3 mg)
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*** Duration of incubation for CBD to bind to Avicel:  about 30 minutes
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*** Washing and Lysis-buffer: 50mM Tris-HCl (pH8.0)
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*** If needed: Elution with 80% ethylen-glycol (EG) or 1/5 Pellet to 4/5 EG (100%) of the overall volume.
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* '''Team Cultivation & Purification:'''
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** Made competent ''P. pastoris'' GS115 cells.
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** Fermentation of ''E. coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012]
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*** Settings: fermenter: Bioengineering NFL22 (7 L), final volume: 6 L, LB-medium with 60 µg/mL chloramphenicol and 300 µg/mL ampicillin added, 37 °C, stirrer increased 2 % if the pO2 got below 30 %, airflow: 5 NL/m. Problem: stirrer cascade did not work at the beginning.
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*** Harvest and centrifugation of cultivation & fermentations 09/14. Store pellet at 4 °C.
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===Sunday September 16th===
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* '''Team Cellulose Binding Domain:'''
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**Colony-PCR of 15 colonies from the [http://partsregistry.org/Part:BBa_K863113 CBDclos(T7)+GFP_His] transformation-plate (biotaq - Armin Nestat's recipe) with the result of a lot of positive clones.
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**Prepared the sequencing of three [http://partsregistry.org/Part:BBa_K863122 const.GFP_His], three [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg] and [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg]
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** Lysis of [http://partsregistry.org/Part:BBa_K863113 CBDcex(T7)+GFP_His] isn't glowing anymore, or never was.
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* '''Team Cultivation & Purification:'''
 
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** Harvest and centrifugation of fermentation of ''E. coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012]. Store pellet at 4 °C.
 
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** Cell disruption via sonification and purification of cultivation of ''E. coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863103 BBa_K863103] via Ni-NTA column.
 
{{Team:Bielefeld/Sponsoren}}
{{Team:Bielefeld/Sponsoren}}

Latest revision as of 21:31, 25 September 2012


Week 20 (09/10 - 09/16/12)

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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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