Team:Bielefeld-Germany/Labjournal/week14

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==Week 14 (07/30 - 08/05/12)==
 
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===Monday July 30th===
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* '''Team Site Directed Mutagenesis:''' Plasmid-isolations of all the bpul- and xccl-mutants.
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* '''Team Cellulose Binding Domain:'''
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**Redesigned the primer-sequences another time giving the CBDs a few additional amino acids from the by Protein-BLAST predicted domain. [http://partsregistry.org/Part:BBa_K863102 CBDcex]: 4 AS N-terminal, 2 C-terminal. [http://partsregistry.org/Part:BBa_K863112 CBDclos]: 2 N-terminal (starting with a natural ATG) and 2 AS C-terminal.
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**Prepared  <partinfo>BBa_K392014</partinfo> for Sequencing
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* '''Team Fungal Laccases:''' Digestion of Tv5 laccase PCR product with Prefix and Suffix ends for cloning in pSB1C3 and restriction of laccase with the overhangs for cloning in shuttle vector. And we did  the PCR on Tv35 with both primer pairs again. This time we lowered the annealing temperatures and got products with both primer pairs.
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*'''Team Cultivation & Purification:'''
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** We made the SDS-Pages for the cultivation from 07/27, but they did not seem to be promising.
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** We started another cultivation of ''E.coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863020 BBa_K863020], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 BBa_K863010], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863015 BBa_K863015] and [https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week1#Monday_April_30th pBpL6].
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::--> Settings: 300mL flasks without baffles, final volume: 60mL, autoinduction medium, 0,25mM CuCl2, 28°C.
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::--> A growth kinetics was recorded every 45 minutes.
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** We decided that we also need a positive control for the next cultivations, to see if our autoinduction medium works. We chose [http://partsregistry.org/Part:BBa_K525710 BBa_K525710].
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** Made a preculture of ''E.coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863020 BBa_K863020], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 BBa_K863010], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863015 BBa_K863015] and [https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week1#Monday_April_30th pBpL6] as well as with [http://partsregistry.org/Part:BBa_K525710 BBa_K525710]. We used ''E.coli'' KRX as negative control.
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===Tuesday July 31st===
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ul {list-style-image:none;}
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* '''Team Site Directed Mutagenesis:''' Plated four colonies per dish of tvel-t243g and tvel-t1161a for plasmid-isolation.
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#bodyContent{
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**Test-restriction of the xccl-mutants showed that no colony was mutated correctly
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    background-color: white;
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**The second site directed mutagenesis of bpul-g2317t could not be rated by test-digestion, since the mutation at 2317 is not because of a illegal restriction site, but a amino acid alternating mutation.
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}
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**Made four bpul-colonies (with both mutations) ready for sequencing
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**plated a four more colonies of xccl (both SDM-sites) for plasmid-isolation
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* '''Team Bacterial Laccases:'''
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:* Ligation of Tv5 laccase with pSB1C3 backbone and purification of Tv35 laccase PCR products.
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:* After our laccases seemed not to get produced we decided to try to express the laccases with constitutive promoters. Therefore we designed primers with the sequences of different promoters from the [Enderson promoter family]. We chose the promoter sequences of the parts [http://partsregistry.org/wiki/index.php/Part:BBa_J23103 BBa_J23103], [http://partsregistry.org/wiki/index.php/Part:BBa_J23103 BBa_J23110] and [http://partsregistry.org/wiki/index.php/Part:BBa_J23103 BBa_J23117] and for all three promoters the RBS [http://partsregistry.org/wiki/index.php/Part:BBa_J23103 BBa_B0034]. The primers were designed that the FW and the RV primers ligate to a short oligonucleotide with overhanging restriction site ends for ''Eco''RI and ''Spe''I. So we don’t have to cut the ligated primers, because the sites should appear with the correct annealing. The goal was to clone the different promoters, the PCR product with the different laccase genes with HIS-Tag with prefix and suffix in pSB1C3 in one ligation step.
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:*Additionally we want to produce the constructs with a new t7 promoter. After our laccases were not expressed we now think that maybe the RBS BBa_B0034, which we changed from originally 5'aAagaggagaaa3' to 5'aGagaggagaaa3' is not or to less recognized from ribosomes in the cells. This is the case, because for designing our T7 promoter overhanging ends for our primers we chose the same sequence as the Bielefeld Team before for example in their part [http://partsregistry.org/Part:BBa_K525710 BBa_K525710]. But another sequence for a T7 promoter, which is described in the Parts Registry was the sequence of the part [http://partsregistry.org/Part:BBa_I719005 BBa_I719005]. So we changed the sequence they used in their part BBa_K525710 from 5'taatacgactcactatagggaAagaggagaaaa 3' to 5’taatacgactcactatagggaGagaggagaaaa 3’ and used this sequence in our primers so the T7 sequence is the one from part [http://partsregistry.org/Part:BBa_I719005 BBa_I719005].
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:: We have a primer pair from last year with the T7 promoter sequence with RBS BBa_B0034. The primers can be annealed to an oligonucleotide. After boiling the primers and cooling down there should be an oligonucleotide with an ''Eco''RI and a ''Pst''I restriction site. We now want to assemble the promoter the laccase genes and the pSB1C3 vector in one step. The pSB1C3 backbone was digested with ''Eco''RI and ''Pst''I, the laccases with ''Xba''I and ''Pst''I, and the promoter has the restriction sites ''Spe''I and ''Eco''RI (we have to digest the promoter with ''Spe''I, because the original restrition site is ''Pst''I).
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* '''Team Fungal Laccases:''' Purification of Tv35 PCR product and digestion for cloning in pSB1C3 backbone.
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<!-- navigator -->
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<div id="nav" class="tabs">
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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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* '''Team Cultivation & Purification:'''
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</ul>
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** Made SDS-Pages of cultivation from 07/30, but they were not promising.
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** Cultivation of ''E.coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005], ''B.halodurans'', [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 BBa_K863010], ''X.campestris'' and [https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week1#Monday_April_30th pBpL6] as well as with [http://partsregistry.org/Part:BBa_K525710 BBa_K525710]. We used ''E.coli'' KRX as negative control.
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::--> general settings: 300 mL flasks without baffles, final volume 60 mL, 30 °C
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::--> special settings:
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:::a) ''E.coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005], [http://partsregistry.org/Part:BBa_K525710 BBa_K525710], ''B.halodurans'', ''X.campestris'' and [https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week1#Monday_April_30th pBpL6] were cultivated in LB media and manually induced after 4 hours with 0,1% rhamnose.
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:::b) ''E.coli'' KRX without plasmid was cultivated with LB medium and induced after 4 hours with 0,1% rhamnose.
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:::c) ''E.coli'' KRX with [http://partsregistry.org/Part:BBa_K525710 BBa_K525710] was cultivated equally to b)
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:::d) ''E.coli'' KRX without plasmid was cultivated with autoinduction medium and 20µg/mL chloramphenicol as well as 100µg/mL ampicillin as a control.
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===Wednesday August 1st===
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</div>
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* '''Team Site Directed Mutagenesis:''' Plasmid-isolation of tvel- and the xccl-colony-dishes.
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</div>
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**Test-restriction with showed correct bands for two tvel and one xccl-colony.
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</html>
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**Made the correct xccl-plasmid ready for sequencing.
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* '''Team Bacterial Laccases:''' With the new T7 promoter we started new assemblies. We now additionally try to make the plasmid with the laccase gene but without His-tag to exclude, that the His6-tag is responsible for no activity of our laccases. We digested our laccase PCR products from ecol without HIS tag for suffix insertion with ''Xba''I and ''Pst''I, boiled and cooled down the joined T7 primer pairs (should have ''Eco''RI and ''Spe''I overhangs after annealing) and digested pSB1C3 backbone with ''Eco''RI and ''Pst''I. If the plan works, the parts anneal to our final plasmid. We did the ligation and transformation of the approach into competent KRX cells.
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* '''Team Cultivation & Purification:'''
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<div style="text-align:justify;">
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** Harvesting and cell disruption via sonification of yesterday's cultivation.
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==Week 14 (07/30 - 08/05/12)==
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===Thursday August 2nd===
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* '''Team Wiki:''' This morning we met for taking a new picture of our group and individual pictures of everyone. Check out our beautiful team members [https://2012.igem.org/Team:Bielefeld-Germany/Students here].
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* '''Team Site Directed Mutagenesis:''' ''pfu''-PCRs; two with the positive tvel-t243g-plasmids as templates and the tvel-t1161a primer-mix and another ''pfu''-PCR with the positve xccl-g2247c-plasmid and the xccl-g3633c primer-mix
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* '''Team Bacterial Laccases:''' There were just red colonies on the plates from the transformation the day before.
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* '''Team Fungal Laccases:''' Ligation of tvel5 and pcil35 in pSB1C3.
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* '''Team Cultivation & Purification:'''
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** Made SDS-Pages from cultivation 07/31.
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===Friday August 3rd===
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* '''Team Site Directed Mutagenesis:'''
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**''Dpn''I-digestion of the ''pfu''-pcr-products (from yesterday). Transformed tevl- and xccl-double-mutants into XL1 Blue and plated them on selection-agar.
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**Sequencing results:
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***Both tthl-g2796a-mutations were successful
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***ecol-g2307a has additional mutations
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* '''Team Cellulose Binding Domain:''' Transformed the plasmids p714 (with the CDBcex domain) and p570 (with the CBDclos domain) we got from the fermentation group of Bielefeld University in KRX an plated them on kanamycin-selection-agar.
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* '''Team Fungal Laccases:''' Transformation of Tv5 and Tv35 in pSB1C3 backbone.
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* '''Team Bacterial Laccases:''' Digest of ecol PCR products with and without HIS-Tag and ligation with pT7 in pSB1C3. Transformation of this ligations and trafo of [http://partsregistry.org/Part:BBa_K525710 BBa_K525710] for Team Cultivation.
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===Saturday August 4th===
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* '''Team Site Directed Mutagenesis:''' Plated four double-mutant-colonies from each transformation (both tvel-mutants and the xccl-mutant on selection-agar for plasmid isolation.
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* '''Team Cellulose Binding Domain:'''
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**The transformation of p570 brought up only a few colonies, so we took a few and plated them again on a selection-agar-dish.
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**Plasmid-isolation of p714
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* '''Team Bacterial Laccases:''' We did the transformation of the ligations from August 1st again.
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* '''Team Cultivation & Purification:'''
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** We prepared precultures of ''E.coli'' KRX without plasmid and ''E.coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005], [https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week1#Monday_April_30th pBpL6] or [http://partsregistry.org/Part:BBa_K525710 BBa_K525710] as positive control.
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===Sunday August 5th===
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* '''Team Site Directed Mutagenesis:''' Plasmid-isolation of the xccl- and tvel-double-mutants.
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**Test-restriction showed two positive plasmids on the xccl-g2247c and xccl-g3633c-mutagensis and no positve tvel.
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**plated two colonies of the ecol-g2307a-mutagenesis and plated them on select-agar for plasmid-isolation.
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**Prepared the two positive double mutants of the xccl-plasmid for sequencing.
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* '''Team Cellulose Binding Domain:'''
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**Transformated BBa_I13522 in KRX and plating it on AMP-selection-agar.
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**Plasmid-isolation of p570
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**PCR of [http://partsregistry.org/Part:BBa_K863103 CBDcex] (417 bp) and [http://partsregistry.org/Part:BBa_K863113 CBDclos] (369 bp) followed by test-gel-electrophoresis showed bands of the correct size and PCR-Clean-up
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**Restriction of PCR-products and pSB1C3_RFP with ''Xba''I and ''Pst''I.
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* '''Team Bacterial Laccases:''' The assemblies don't work in one step by now so we started to clone one part after another in pSb1C3 backbone. For this reason we additionally want to ligate the ecol gene without any promoter in pSB1C3 backbone and in the next step do a prefix insertion with the promoter fragment. Therefore we did the restriction of ecol and ecol_HIS PCR products, the ligation in pSB1C3 and the transformation.
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* '''Team Cultivation & Purification:'''
 
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** We made another flask cultivation of ''E.coli'' KRX without plasmid and with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005], [https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week1#Monday_April_30th pBpL6] and [http://partsregistry.org/Part:BBa_K525710 BBa_K525710] as positive control.
 
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::--> general settings: 250mL flasks without baffles, final volume: 50mL, LB medium
 
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::--> additional settings:
 
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::: a) 2 flasks of each culture were inducted with 0,1% rhamnose after 4 hours of cultivation
 
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::: b) 2 flasks of each culture were not induced.
 
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::-->We recorded the growth kinetics once per hour.
 
{{Team:Bielefeld/Sponsoren}}
{{Team:Bielefeld/Sponsoren}}

Latest revision as of 21:24, 25 September 2012


Week 14 (07/30 - 08/05/12)

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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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