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| | {{Team:Bielefeld/Head}} | | {{Team:Bielefeld/Head}} |
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| - | <div style="text-align:justify;">
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| - | ==Week 15 (08/06 - 08/12/12)== | + | <html> |
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| - | ===Monday August 6th===
| + | ul {list-style-image:none;} |
| - | * '''Team Site Directed Mutagenesis:''' Plasmid-isolation of two more ecol-g2307a colonies
| + | #bodyContent{ |
| - | * '''Team Cellulose Binding Domain:''' Plated one colony of Bba_I13522
| + | background-color: white; |
| - | **Dephosphorylation of pSB1C3 Backbone
| + | } |
| - | **Ligation of CBDcex+pSB1C3 and CBDclos+pSB1C3
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| - | **Transformed in KRX and plated on CM-selection-agar
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| - | * '''Team Bacterial Laccases:'''
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| - | ** No positive colonies after transformation of our assemblies from August 1st, but we realized that the primers we used for making the promoter parts can’t ligate with our insert an backbone because the primers are dephosphorylated and the plasmid backbone is dephosphorylated, too. Much effort in a mission which can't work but at least we know now why it doesn't work.
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| - | :* Picking positive colonies from transformation of ecol and ecol_HIS in pSB1C3 for plasmid isolation.
| + | </style> |
| - | * '''Team Fungal Laccases:''' Plating positive colonies from cloning of tvel5 in pSb1C3 backbone.
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| | | | |
| - | ===Tuesday August 7th=== | + | <!-- navigator --> |
| - | * '''Team Bacterial Laccases:''' Plasmid isolation and control restriction of ecol and ecol_HIS in pSB1C3 showed correct fragment sizes in agarose gel. So we did a digest for prefix insertion of the new T7 promoter.
| + | <div id="nav" class="tabs"> |
| - | * '''Team Site Directed Mutagenesis:''' Test-digestion of the two ecol-g2307a-mutants showed that one has lost the restriction-site. Prepared that one for sequencing.
| + | <div class="scroller"> |
| | + | <ul style="list-style-type:none"> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/Starting"><strong>Prologue</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week1"><strong>Week 1</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week2"><strong>Week 2</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week3"><strong>Week 3</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week4"><strong>Week 4</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week5"><strong>Week 5</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week6"><strong>Week 6</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week7"><strong>Week 7</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week8"><strong>Week 8</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week9"><strong>Week 9</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week10"><strong>Week 10</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week11"><strong>Week 11</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week12"><strong>Week 12</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week13"><strong>Week 13</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week14"><strong>Week 14</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week15"><strong>Week 15</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week16"><strong>Week 16</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week17"><strong>Week 17</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week18"><strong>Week 18</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week19"><strong>Week 19</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week20"><strong>Week 20</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week21"><strong>Week 21</strong></a></li> |
| | + | <li><a href="https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week22"><strong>Week 22</strong></a></li> |
| | | | |
| - | ===Wednesday August 8th===
| + | </ul> |
| - | * '''Team Bacterial Laccases:''' We dephosphorylated the digested the plasmids from day before and phosphorylated the promoter parts. After that we ligated the two parts and transformated the products into KRX electrocompetent cells.
| + | |
| - | * '''Team Fungal Laccases:''' Plasmid isolation of tvel5 laccase in pSB1C3 backbone.
| + | |
| - | * '''Team Site Directed Mutagenesis:''' Made PCRs on tvel-t143g-mutants with Tv_lac10.P.FW and Tv_lac10.S.RV primers, with products of 1.6 kbp when there should be about 4.0 kbp.
| + | |
| | | | |
| - | ===Thursday August 9th===
| + | </div> |
| - | * '''Team Fungal Laccases:''' Again: Ligation of tvel35 in pSB1C3 backbone.
| + | </div> |
| - | * '''Team Cultivation & Purification:'''
| + | </html> |
| - | ** We got ''E.coli'' KRX with our own BioBrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] in pSB1C3 like the rest.
| + | |
| - | ** We discussed if we did not produce anything because of the toxicity of our protein, which may reduce the plasmid stability. We searched for maximal used concentration of chloramphenicol and found out that concentrations up to 170µg/mL were used. Therefore we decided to start a new flask cultivation with concentrations of chloramphenicol varying in 5 steps from 20µg/mL to 170µg/mL. Today we made the precultures of ''E.coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005] as well as ''B.halodurans'', ''X.campestris'' and ''?T.thermophilus?''. We used ''E.coli'' KRX as negative and [http://partsregistry.org/Part:BBa_K525710 BBa_K525710] as positive control.
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| - | * '''Team Site Directed Mutagenesis:''' Results from the Sequencing arrived:
| + | |
| - | **One bpul-plasmid is positive on both mutations
| + | |
| - | **All Xccl-mutants are negative (there even was a part of 900 bp gone missing!)
| + | |
| - | **plated six more colonies of the xccl-g3633c for plasmid-isolation
| + | |
| - | **PCR with the original tvel10-plasmid and the Prefix/Suffix-primers, showed no product at all.
| + | |
| | | | |
| - | ===Friday August 10th=== | + | <div style="text-align:justify;"> |
| - | * '''Team Bacterial Laccases:'''
| + | |
| - | :* Again we did the digest of our new T7 promoter part and the ligation in pSB1C3 backbone with ecol ORF PCR products with and without HIS tag. After that we transformed the ligations in pSB1C3. Additionally we did the same with promoter J23110 instead of T7 promoter.
| + | |
| - | :* We did PCR on [http://partsregistry.org/Part:BBa_K863020 BBa_K863020] with the primers B.halo_FW and B.halo_RV for cloning the gene in pSB1C3 backbone without promoter and HIS tag.
| + | |
| - | :* We ligated the digested pSB1C3 plasmids with ecol and ecol_HIS with the new pT7 promoter and pSB1C3 backbone and transformed the approach in KRX. | + | |
| | | | |
| - | *'''Team Cultivation & Purification:'''
| + | ==Week 15 (08/06 - 08/12/12)== |
| - | :* Flask cultivation of ''E.coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005]; ''B.halodurans'', ''X.campestris'' and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863010 BBa_K863010] to test various concentrations of chloramphenicol.. We used ''E.coli'' KRX as negative and ''E.coli'' KRX with [http://partsregistry.org/Part:BBa_K525710 BBa_K525710] as positive control.
| + | |
| - | :--> Settings: 100mL flasks without baffles, final volume: 10mL, autoinduction medium with 20/ 57,5/ 95/ 132,5/ 170µg/mL chloramphenicol, 37°C, 120rpm, double determination.
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| | | | |
| - | ===Saturday August 11th===
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| - | * '''Team Bacterial Laccases:'''
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| - | :* Colony PCRs showed no bands. So we transformed the ligations from 10.08. again.
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| - | :* We did the PCRs of the laccase genes ecol, bpul, bhal and lthl again. We used the …_FW / …_RV primers and the …_FW / …_RV_HIS primers of the different genes. Digestion of this PCR products and ligation with pT7 or promoter J23110 and the pSB1C3 plasmid backbone.
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| | | | |
| - | ===Sunday August 12th===
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| - | * '''Team Bacterial Laccases:''' Cleanup from agarose gel of the PCR products from the day before. After that we did control restriction and got bands for ecol with J23110 promoter and with the new pT7. So we sent this plasmids for sequencing.
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| | {{Team:Bielefeld/Sponsoren}} | | {{Team:Bielefeld/Sponsoren}} |
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