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| | ==Week 8 (06/18 - 06/24/12)== | | ==Week 8 (06/18 - 06/24/12)== |
| - | === weekly seminar ===
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| - | * Julia V. presents the database to have an easier overview over the labwork, the biobricks and as a digital lab diary.
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| - | * the last changes have been implemented into the press release, it is about time to let the world know about our project.
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| - | * Everyone please keep the iGEM deadlines in mind.
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| - | * Our advisor Timo presents: iGEM 101: Introduction to the wiki.
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| - | * Julia S is presenting her progesses in designing the ''Pichia pastoris'' shuttle vector.
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| - | * Modelling: Julia V. is thinking about how expression rates and promoter activity can be implemented into the model. Sebastian tries to model a sewage treatment plant.
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| - | ===Monday June 18th===
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| - | * '''Team Cloning of Bacterial Laccases:'''
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| - | **We started Colony PCRs on the colonies from June 15th transformation and picked positive colonies to plate them for plasmid isolation. Sadly we just had positive clones for tthl(T7)_His and not for bpul(T7)_His.
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| - | * '''Team Site Directed Mutagenesis:'''
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| - | ** Reviewed all Assembly-Standards and made a list of all illegal restriction-sites:
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| - | **''Eco''RI, ''Not''I, ''Pst''I, ''Spe''I, ''Xba''I (Silver), ''Age''I, ''Ngo''MIV (Freiburg), ''Bam''HI, ''Bgl''II, ''Xho''I (Berkeley)
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| - | ** Decided to not care for restriction-sites of the Berkeley-assembly (even if it is a great assembly for protein-fusion), because the used Vector (pSB1C3) already has two ''Xho''I-restriction-sites
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| - | *'''Team Fungal and Plant Laccases'''
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| - | ** We have not received any reply to our e-mail. Team meeting to discuss the further way forward. We decided to send a second wave of e-mails to the different working groups, try to call the working groups(if possible) and to look for strains of the corresponding and published sequences in additional straincollections.
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| - | ===Tuesday June 19th===
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| - | * '''Team Wiki''':
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| - | ** While using the lab journal more frequently there came up some questions.
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| - | ** How detailed do we plan to write our lab journal entries?
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| - | ** Do we want to write in keywords or explain everything in full sentences?
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| - | ** Do we want to note every little detail about every successful or unsuccessful experiment or just the main important aspect?
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| - | ** We discussed, sighted some former iGEM team wikis and decided:
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| - | *** each team is responsible for their own lab journal entries
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| - | *** we divide our lab journal in weeks <span style="text-decoration: underline;">and</span> days to prevent it from looking too chaotic.
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| - | *** the texts are supposed to state which team is writing, which experiment has been done and what the main aspects were. Also we will write about successful experiments, as well as problems and solutions we came up with. If possible links to protocols with further information shall be created.
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| - | ===Wednesday June 20th===
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| - | * '''Team Site Directed Mutagenesis:'''
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| - | ** Imported sequences of most of the used bacterial Laccases into Clonemanager and analysed their restriction-sites:
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| - | ** bhal has no illegal restriction-sites
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| - | ** ecol has one ''Ngo''MIV-restriction-site
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| - | ** bpul has one ''Xba''I-Restriction-site and one mutation two ''Age''I- and two ''Ngo''MIV-restriction-sites (Decided to delete the Freiburg-restriction-sites would take too much time)
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| - | ** tthl has one ''Pst''I-restriction-site and two ''Ngo''MIV-restriction-sites (Decided to delete the Freiburg-restriction-sites would take too much time)
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| - | ** xccl has two ''Pst''I, one ''Age''I and seven ''Ngo''MIV-restriction-sites (Decided not to change the ''Ngo''MIV-sites, since to mutate seven would take too much time)
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| - | * '''Team Modeling:'''
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| - | ** Finding out, that the "normal" Michaelis-Menten kinetic isn't the right kinetic to model our situation, because therefor you need a high and steady state concentration of the substrates. We have low concentrations and not really study state. We found a transformed equation.
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| - | ===Thursday June 21st===
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| - | * '''Team Cloning of Bacterial Laccases:'''
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| - | ** Plasmid isolation and control digest with ''Not''I on tthl(T7)_His and luckily this time the bands were where they should be. Again and again we did transformation of ligation with bpul(T7)_His laccase in pSB1C3 backbone..all fingers are crossed that this time we have colonies with the correct plasmid.
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| - | * '''Team Activity Tests''' and '''Team Immobilization:'''
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| - | ** After all this characterizing we feel so much closer to our ''T. versicolor'' laccase that its about time to make some activity test under immobilized conditions. So now we are cooperating with Team Immobilization. We have thought about many ways how to immobilize the laccase and decided to give Silica Beads the first try. Check the Immobilization Team´s protocol for further information. Our main problem was how to measure the samples with all those beads in it. Tecan will probably be confused and give us some false values due to the beads that are disturbing its laser. So we need a way to get the beads out (and thus also stop the reaction) at a very precise point of time. Centrifugation wasn´t an option because it would simply take too long and not stop the reaction exactly in the second we want. While checking the internet for solutions we found Multi-Well Membrane-Bottom Filter Plates. Those are supposed to work in a similar way then our regular plates which we used for the Tecan but furthermore those plates contain a membrane that sieve the liquids through the filter when centrifugated. Thus the beads are separated and the ABTS-Buffer solution can me analyzed at 420 nm for oxidized ABTS. The plates will need a while before they arrive here at the CeBiTec, so we decided to first find out what the optimal amount of beads is and whether the beads might also bind ABTS (see lab journal Team Immobilization).
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| - | *'''Team Immobilization:'''
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| - | **So after a lot of reading and discussion, we decided to try immobilization using beads. Since silica beads were already available in our lab (from last year’s team), we decided to give them a try. The first challenge was to find out a convenient ratio of beads/laccase. According to the protocol of last year’s team, the ratio 1:1000 was used (1000mg beads/ 1 mg protein). Therefore we decided to try the ratios 1:500, 1:1000 and 1:1500. We prepared different buffers: [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Immobilization HBSS buffer], [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Immobilization Recrystallization buffer] both of which were used with silica beads; in addition to [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Immobilization Britton-Robinson Buffer], which was mentioned in publications as the best buffer for laccase immobilization. Laccases from TVEL0 were incubated with silica beads and different buffers at room temperature for 4hours on a rotator. After that, we collected the supernatants and delivered them to the team “Activity test” and waited for the good news.
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| - | ===Friday June 22nd===
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| - | * '''Team Cloning of Bacterial Laccases:'''
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| - | **Because our PCR didn't work on the boiled lyophilized cells we used [http://www.carlroth.com/catalogue/catalogue.do;jsessionid=2E5F0AF60BF1F28909D8475CF0053386?favOid=00000000000180e500070023&act=showBookmark&lang=de-de&market=DE CASO Medium] for cultivation of ''S. griseus'' and ''S. lavendulae''.
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| - | *'''Team Immobilization:'''
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| - | **Unfortunately, the activity test results weren’t promising. According to publications, immobilization via covalent binding is the most widely used method. Silica dioxide bead offer only an adsorption of laccases. Therefore, we decided to order CPC-(controlled pore carrier) silica beads, to which laccases covalently bind, especially that we found some papers with protocols and activity tests proving their efficiency.
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| - | ===Saturday June 23rd===
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| - | * '''Team Cloning of Bacterial Laccases:'''
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| - | ** The cultured ''S. griseus'' and ''S. lavendulae'' bacterials has been centrifuged at 13.000 rpm for 5 minutes. After this step we ribolyzed the pellet in 1 ml TE-Puffer and set a PCR reaction after. But we still haven't had any results.
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| - | ** Colony PCRs on the transformations with plasmid with bpul(T7)_His and plating positive colonies.
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| - | * '''Team Database:'''
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| - | ** In the last wekk we finished the tabels Eppi, User, Sequencing and BioBrick.
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| - | ** We have a problem with the search function, we use the false SQL statement and allready deleted entries are shown.
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| - | ** In the next week we have to write a new statement.
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| - | ===Sunday June 24th===
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| - | *'''Team Cloning of Bacterial Laccases''':
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| - | ** We isolated plasmids and did control digests with ''Not''I. We finally had a positive restriction digest for bupl(T7)_His. So we prepared this plasmids and the plasmid tthl(T7)_His which we isolated some days before for sequencing.
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| | {{Team:Bielefeld/Sponsoren}} | | {{Team:Bielefeld/Sponsoren}} |
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