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| | ==Week 12 (07/16 - 07/22/12)== | | ==Week 12 (07/16 - 07/22/12)== |
| - | === weekly seminar ===
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| - | * For our sponsoring we sent all the treaties, to all our sponsors so far.
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| - | * Julia V. will organize a guided tour to a sewage treatment plant.
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| - | * The summer school was a great success and all the pupils appreciated our organization and the experiments.
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| - | * Julia V. will be responsible for the wiki design from now on.
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| - | * A big question appeared: How can we put our database on the wiki?
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| - | * We will take new team photos next week.
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| - | * For the safety/security everyone has to read the safety regulations regarding the organisms and chemicals.
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| - | * Next week there will be a short presentation about all used organisms and their laccases
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| - | * Agatha and Saskia will create templates for our webpages.
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| - | * Nadine will gather information about places to stay in Amsterdam and how to get there.
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| - | * Everyone has a passport?
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| - | * Gabi will present the judging criteria next week.
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| - | * Julia is looking for a BioBrick, which we could use and improve.
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| - | * Isabel created a poster for the conference in Munich, everyone has to review it.
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| - |
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| - | ===Monday July 16th===
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| - | *'''Team Fungal and Plant Laccases:'''
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| - | **After we forgot to delete the signal peptide sequences, which are present in the fungal laccases we designed new forward primers for the laccases tvel5, tvel10, tvel13, tvel20 and pcil35 with the overhanging ends for cloning in our shuttle vector. ''Trametes'' laccases have a signal sequence after the start codon. This signal peptide we now delete by taking the first 20 bases after this sequence in our FW primers.
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| - | **Our plants had a great time during the last weeks in the climate chamber. So today it was time for them to donate their seeds for RNA isolation, cDNA synthesis and a PCR (check [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/molecular_genetics#Arabidopsis_thaliana:_Total_RNA_Isolation protocols]). We ran an additional sample with actin primers as a positive control. However both samples did not show any bands. Maybe the high salt concentration in our sample was responsible or the laccase concentration in the 1:10 diluted cDNA was too low. We will do some washing and try again.
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| - | * '''Team Cultivation & Purification''':
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| - | ** We searched for some information for the best cultivation conditions in the internet. We found an interesting report of the [http://www.dbu.de/OPAC/ab/DBU-Abschlussbericht-AZ-13191.pdf Deutsche Bundesstiftung Umwelt (DBU)] containing some interesting facts about different laccases as for example that the bacterial laccases are toxic to the bacterias, so that the production could be better under oxygen limitation and reduced temperature. Based on this article we decided to test flask with and without baffles and different temperatures.
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| - | ** We prepared the basic media and solutions we will need in the lab.
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| - | ** Note: All following BioBricks are cloned into pSB1C3 and therefore cultivated with 20 µg/mL chloramphenicol unless otherwise specified! Cultivations of ''E. coli'' KRX without plasmid will be performed without antibiotics.
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| - | ===Tuesday July 17th===
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| - | * '''Team Site Directed Mutagenesis:''' Made Clone Manager files for the ''Trametes versicolor'' laccase plasmids we got send and analyzed them:
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| - | **tvel5:
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| - | ***one silent mutation (at 154 bp) and one amino acid alternating mutation at 227 bp (G for A, replacing D with N), a third was claimed to be at 1559 but sequencing showed it wasn’t.
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| - | ***No illegal restriction-site for the Silver-assembly, but three Freiburg-assembly restriction-sites: two ''Ngo''MIV- and one ''Age''I-restriction-sites
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| - | **tvel10:
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| - | ***eight silent mutations 171, 444, 1020, 1173, 1239, 1443, 1485 & 1503 bp
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| - | ***Two amino acid alternating mutations (1048 bp C for T, replacing F with L and 1078 bp A for G, replacing D with N)
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| - | ***Two ''Pst''I-restriction-sites (One in the signaling-sequence at 7 bp and one at 1160) and one ''Spe''I-restriction-site at 241 bp
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| - | ***One Freiburg-assembly restriction-site (''Age''I at 912 bp)
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| - | **tvel13:
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| - | ***56 silent mutations
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| - | ***Three amino acid alternating mutations and one whole codon is missing at the very end
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| - | ***One ''Eco''RI- and one ''Pst''I-restriction-site
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| - | ***Two Freiburg-assembly restriction-sites (two ''Ngo''MIV-sites)
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| - | **tvel20:
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| - | ***about 32 silent mutations, three amino acid alternating mutations and four Freiburg-assembly restriction-sites (one ''AgeI'', three ''Ngo''MIV)
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| - | **pcil35:
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| - | ***no illegal restriction-site
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| - | * '''Team Cultivation & Purification:'''
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| - | ** We prepared our first precultures of ''E. coli'' KRX as negative control and ''E. coli'' KRX containing [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_K863015 BBa_K863015] and [https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week1#Monday_April_30th pBpL6].
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| - | ** Note: We will cultivate ''E. coli'' KRX with [https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week1#Monday_April_30th pBpL6] until we will get the laccase ORF with T7 promotor and His tag in the same pSB1C3 vector as the other BioBricks. To cultivations of ''E. coli'' KRX with pBpL6 we always will add 100 µg/mL ampicillin.
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| - | ===Wednesday July 18th===
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| - | * '''Team Site Directed Mutagenesis:''' Made Primer-Mixes for the bacterial laccases. Set Pre-culture of XL1 blue. Got everything ready for Lab work.
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| - | * '''Team Cloning of Bacterial Laccases''':
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| - | :* We diluted our chromosomal DNA to a concentration of 20 ng/µL, since the volume we got was to low for doing many PCRs and did again a PCR reaction. This time ''S. goettingen'' laccase DNA could be identified but not ''S. tuebingen''. The reaction conditions were the same so we were surprised because ''S. tuebingen'' didn`t work.
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| - | * '''Team Fungal and Plant Laccases''': Some of the ordered parts from the Parts Registry arrived and we plated the biobricks [http://partsregistry.org/Part:BBa_K500000 BBa_K500000], [http://partsregistry.org/Part:BBa_K500001 BBa_K500001], [http://partsregistry.org/Part:BBa_K500002 BBa_K500002], [http://partsregistry.org/Part:BBa_K500003 BBa_K500003] and [http://partsregistry.org/Part:BBa_K392014 BBa_K392014]. Above all we are interested in [http://partsregistry.org/Part:BBa_K500002 BBa_K500002] because it’s a codon optimized laccase from ''Trametes versicolor'' and we want to use this laccase in our ''P. pastoris'' shuttle vector and characterize it.
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| - | *'''Team Cultivation & Purification''':
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| - | ** Today we performed our first flask cultivation. We cultivated ''E. coli'' KRX with [http://partsregistry.org/Part:BBa_K500005 BBa_K500005], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863020 BBa_K863020], [https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week1#Monday_April_30th pBpL6] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863015 BBa_K863015] and as negative control we used ''E. coli'' KRX.
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| - | *** Settings: We used 300 mL flasks without baffles, final volume: 60 mL, [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium], 30/37 °C, durance: 24 hours
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| - | ** Found out that we had a mixed culture of ''E. coli'' KRX with [https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week1#Monday_April_30th pBpL6], because it grew on chloramphenicol, but has only an ampicilline resistance. So we could not use this culture.
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| - | ===Thursday July 19th===
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| - | * '''Team Site Directed Mutagenesis:'''
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| - | **Made electrocompetent XL1 blue cells. Made ''pfu''-PCR of the bpul-plasmid with bpul-a2883t primer-mix, the ecol-plasmid with ecol-g2307a primer-mix, the xccl-plasmid with the xccl-g2247c primer-mix and tthl-plasmid with the tthl-g2796a primer-mix. Digested the template with ''Dpn''I.
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| - | ** Generated Primers for silent mutations of tvel10 illegal restriction-sites:
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| - | ***the illegal ''Spe''I will be deleted by changing acT to ggA (Threonine) at 243 bp of the know sequence
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| - | ***the illegal ''Pst''I will be deleted by changing ccT to ccA (Proline) at 1161 bp of the known sequence
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| - | ***one illegal restriction site in the signaling-sequence can not be mutated, since it is to close to the beginning of the known sequence (7 bp) and also it is not essencial, because the gene will be used without the signaling sequence
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| - | * '''Team Cellulose Binding Domain:'''
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| - | **Gathered some information about carbohydrate binding domain X2 (which is a more common domain in the organisms we handle than a cellulose Binding Domain).
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| - | **Used NCBI Nucleotid BLAST on <partinfo>BBa_K392014</partinfo> (the Cellulose-binding motif from ''C. josui'' Xyn10A gene) and it 100% fits to 1526 bp to 2608bp of AB041993.1 (''Clostridium josui'' xynA gene for xylanase A).
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| - | **Used NCBI Protein-BLAST on AB041993.1 and found one cellulose binding domain and one carbohydrate binding domain within the protein.:
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| - | ***pfam02018: CBM_4_9 (Carbohydrate binding domain) from AS 193 to AS 332 - corresponds to 1088 bp - 1507 bp in the gene and is not in the coding sequence of <partinfo>BBa_K392014</partinfo>.
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| - | ***cd09619: CBM9_like_4 (DOMON-like type 9 carbohydrate binding module) from AS 716 to AS 887 - corresponds to 2657 bp to 3172 bp in the gene and is also not in coding sequence of <partinfo>K392014</partinfo>.
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| - | ***Given sequence of <partinfo>BBa_K392014</partinfo> (1589bp to 2590bp) does corresponds to the sequence of the Glycosyl hydrolase 1589 bp to 2590 bp and is not one of the binding motifs. The whole review can be read at the [http://partsregistry.org/Part:BBa_K392014:Experience Experience-page of BBa_K392014].
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| - | * '''Team Cloning of Bacterial Laccases:'''
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| - | :*''S. goettingen'' and ''S. tuebingen'' DNA were cleaned up and we done an enzymatic digestion to ligate it into the pSB1C3 vector. We did the digestion with EcoRI and SpeI.
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| - | * '''Team Fungal and Plant Laccases:''' Colonies from plated BioBricks from 18.07 were spread on nutrient agar for plasmid isolation.
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| - | * '''Team Cultivation & Purification:'''
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| - | ** The cultures of 07/18 were centrifugated, cells were disrupted via sonification in the [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#special_buffers_for_resuspending_the_cell_pellet special buffer] for each laccase and after another centrifugation the supernatant was given to the activity test team.
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| - | ** Made precultures analogous to them on 07/17.
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| - | ===Friday July 20th===
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| - | * '''Team Modeling:''' We need a contact to a clarification plant to get information about clarification plant itself and perhaps to proof our cleaner with real probes. therefor we are calling Mr. Bülter form the clarification plant Schloß Holte (near Bielefeld) and he invited us to present our project.
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| - | * '''Team Site Directed Mutagenesis:'''
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| - | **Transformation of XL1 Blue with the PCR products (see day before)
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| - | * '''Team Cellulose Binding Domain:'''
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| - | **Used Protein-BLAST on the translated sequence of the ''Clostridium cellulovorans'' cellulose binding protein, the ''Bacillus halodurans'' strain Cochin chitinase GU481106.1 and chitin-binding protein [''Bacillus halodurans C-125''] BAB05022.1, to get more information about the predicted sequences of Carbohydrate binding domains.
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| - | **Looked up possible linkers in the Parts Registry:
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| - | ***2 aa GS linker: [http://partsregistry.org/Part:BBa_J18920 BBa_J18920] [GS]
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| - | ***10 aa [GS]x linker: <partinfo>BBa_J18922</partinfo> [GSGSGSGSGS]
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| - | ***15 aa flexible glycine-serine protein domain linker; Freiburg standard 1 Star: <partinfo>BBa_K157013</partinfo> [GGGGSGGGGSGGGGS]
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| - | ***10 aa flexible protein domain linker 1 Star: <partinfo>BBa_K105012</partinfo>: [GENLYFQSGG]
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| - | **Made a few possible primers for [http://partsregistry.org/Part:BBa_K863112 CBDclos] with T7 and RBS (<partinfo>BBa_K525998</partinfo>), a Freiburg-Suffix and possible linkers
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| - | **Wrote an E-Mail to Jun.Prof Thorsten Seidel (FRET-Expert of Bielefeld University) asking him about the linkers they use for fusing GFP for FRET.
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| - | * '''Team Fungal and Plant Laccases:''' Isolating the plasmids [http://partsregistry.org/Part:BBa_K500000 BBa_K500000], [http://partsregistry.org/Part:BBa_K500001 BBa_K500001], [http://partsregistry.org/Part:BBa_K500002 BBa_K500002], [http://partsregistry.org/Part:BBa_K500003 BBa_K500003] and [http://partsregistry.org/Part:BBa_K392014 BBa_K392014]. To be sure that in every plasmid contains the correct part we made a control restriction with ''Not''I. It showed that all parts are on the correct hight in agarose gel.
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| - | * '''Team Cultivation & Purification:'''
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| - | ** Another flask cultivation was made analogous to the one on 07/18 but at 23 °C.
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| - | ===Saturday July 21st===
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| - | * '''Team Cellulose Binding Domain:'''
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| - | **Made primers for [http://partsregistry.org/Part:BBa_K863121 GFP_His] (using [http://partsregistry.org/Part:BBa_I13522 BBa_I13522] as template)adding a C-termial His6tag for easy extraction
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| - | **Jun.Prof Thorsten Seidel answered to the mail of yesterday and said that GFPs are easy to fuse and that they use linkers with four AS:
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| - | ***ccg gtc gcc acc upstream of the GFP
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| - | ***gaa agc ggc cgc downstream of the GFP; Which is will work fine, even with a proline in it's sequence.
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| - | **We decided to use four linking AS also and chose to add a C-terminal Glycine-Serine-Linker ([http://partsregistry.org/Part:BBa_J18920 BBa_J18920]) to the cellulose binding domain, which would be four linking AS with the Freiburg-scar (Threonine-Glycine).
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| - | * '''Team Cloning of Bacterial Laccases''':
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| - | :* Since our PCRs didn't work and the ''Streptomyces'' laccases have more then 70% GC content we changed the dNTP concentration to 10 mM per base. Our gelelectrophoresis showed us that again ''S. goettingen'' has a product in correct length so we did again a PCR with same conditions. The rest of this product was cutted from an agarose gel and set ligation with the pSB1C3 vector and transformation. Because the isolated DNA concentration was to low the transformation showed no positive results.
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| - | * '''Team Cultivation & Purification:'''
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| - | ** Culture from 07/20 was harvested and the cells were disrupted via sonification.
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| - | ===Sunday July 22nd===
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| - | * '''Team Cellulose Binding Domain:'''
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| - | **We decided to use the exact sequence of [http://partsregistry.org/Part:BBa_J18920 BBa_J18920] (GGCAGC) of the linker in the partsregistry.
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| - | **Checked the CBDs we had access to for illegal restriction sites and found none, not even ''Ngo''MIV or ''Age''I. So we decided to use a Freiburg-Assembly to build the CBD-GFP fusion protein.
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| | {{Team:Bielefeld/Sponsoren}} | | {{Team:Bielefeld/Sponsoren}} |
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