Team:Bielefeld-Germany/Labjournal/week12

Week 12 (07/16 - 07/22/12)

Monday July 16th

• Team A. thaliana laccase: 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 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.
• Team Fungal Laccases: After we forgot to delete the signal peptide sequences, which are present in the fungal laccases we designed new forward primers for the laccases Tv5, Tv10, Tv13, Tv20 and Pc35 with the overhanging ends for cloning in our shuttle vector. Trametes laccases have a signal sequence after the start codon. This signal peptide we deleted by taking the first 20 bases after this sequence in our FW primers.

Tuesday July 17th

• Team Site Directed Mutagenesis: Made Clone Manager files for the Trametes versicolor-laccase-plasmids we got send and analysed them:
  • T.v.5:
    • 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.
    • No illegal restriction-site for the Silver-assembly, but three Freiburg-assembly restriction-sites: two NgoMIV- and one AgeI-restriction-sites
  • T.v.10:
    • eight silent mutations 171, 444, 1020, 1173, 1239, 1443, 1485 & 1503 bp
    • Two amino acid alternating mutations (1048 bp C for T, replacing F with L and 1078 bp A for G, replacing D with N)
    • Two PstI-restriction-sites (One in the signaling-sequence at 7 bp and one at 1160) and one SpeI-restriction-site at 241 bp
    • One Freiburg-assembly restriction-site (AgeI at 912 bp)
  • T.v.13:
    • 56 silent mutations
    • Three amino acid alternating mutations and one whole codon is missing at the very end
    • One EcoRI- and one PstI-restriction-site
    • Two Freiburg-assembly restriction-sites (two NgoMIV-sites)
  • T.v.20:
    • about 32 silent mutations, three amino acid alternating mutations and four Freiburg-assembly restriction-sites (one AgeI, three NgoMIV)
  • P.c.35:
    • no illegal restriction-site

Wednesday July 18th

• Team Site Directed Mutagenesis: Made Primer-Mixes for the bacterial laccases. Got everything ready for Lab work.
• Team Bacterial Laccases: We diluted our chromosomal DNA to a concentration of 20ng/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.
• Team Fungal 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.
• Team Cultivation & Purification:
  • Today we performed our second flask cultivation. We cultivated E.coli KRX with laccases from E.coli, B.halodurans, B.pumilus and X.campestris and as negative control we usedE.coli KRX without plasmid. This time we changed some settings. We used: flask without baffles (300mL),autoinduction medium with 20µg/mL chloramphenicol added, final volume of 60mL, 30/37°C, durance: 24 hours
  • Found out that we had a mixed culture of B.pumilus', because it growth on chloramphenicol, but has only an ampicilline resistance. So we could not use this cultur.

Thursday July 19th

• Team Site Directed Mutagenesis: pfu-PCR and DpnI restriction of the SDM to mutate the B.pumilus laccase plasmid at 2883, the E.coli CueO plasmid at 2307, the X.campestris at 2247 and T.thermophilus plasmid at 2796.
  • **Generated Primers for silent mutations of ‘’T.v.’’10 illegal restriction-sites:
    • illegal SpeI at 243 bp changing acT to ggA (Threonine)
    • illegal PstI at 1161 bp ccT to ccA (Proline)
    • one illegal restriction site in the signaling-sequence will not be mutated, since it is to close to the beginning of the known sequence (7 bp)and the gene will be used without the signaling sequence
• Team Cellulose Binding Domain: Got info about Carbohydrate binding domain X2 (which is a more common domain in the organisms we handle than a Cellulose Binding Domain.)
  • Nucleotid BLASTed BBa_K392014 (Cellulose-binding motif from C. josui Xyn10A gene) -> 1526bp to 2608bp of AB041993.1 (Clostridium josui xynA gene for xylanase A)
  • Protein-BLASTed AB041993.1 and found got one cellulose binding site and one carbohydrate binding domain.:
    • pfam02018: CBM_4_9 (Carbohydrate binding domain) from AS 193 to AS 332 - corresponds to 1088 - 1507 in Gene and is not in the coding sequence of BBa_K392014
    • cd09619: CBM9_like_4 (DOMON-like type 9 carbohydrate binding module) from AS 716 to AS 887 - corresponds to 2657bp to 3172bp in the gene and is also not in coding sequence of BBa_K392014
    • Given sequence of BBa_K392014 (1589bp to 2590bp) does corresponds to the sequence of the Glycosyl hydrolase 1589bp to 2590bp and not one of the binding motifs
• Team Bacterial Laccases: 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.
• Team Fungal Laccases: Colonies from plated biobricks from 18.07 were spread on nutrient agar for plasmid isolation.
• Team Cultivation & Purification:
  • The cultures were centrifugated, cells were disrupted via sonification in the special buffer for each laccase and after another centrifugation the supernatant was given to the activity test team.

Friday July 20th

• 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.
• Team Site Directed Mutagenesis: Transformation of XL1 Blue with the PCR products (see day before)
• Team Cellulose Binding Domain: Protein-BLASTed the Proteinsequence of Clostridium cellulovorans cellulose binding protein, Bacillus halodurans strain Cochin chitinase GU481106.1 and chitin-binding protein [Bacillus halodurans C-125] BAB05022.1, to get the predicted sequences of CBDs
  • Looked up possible linkers in the Parts Registry:
    • 2 aa GS linker: [http://partsregistry.org/Part:BBa_J18920 BBa_J18920] [GS]
    • 10 aa [GS]x linker: BBa_J18922 [GSGSGSGSGS]
    • 15 aa flexible glycine-serine protein domain linker; Freiburg standard 1 Star: BBa_K157013 [GGGGSGGGGSGGGGS]
    • 10 aa flexible protein domain linker 1 Star: BBa_K105012: [GENLYFQSGG]
  • Made a few possible primers for CBDclos with T7 and RBS (BBa_K525998), a Freiburg-Suffix and possible linkers
  • Wrote an E-Mail to Jun.Prof Thorsten Seidel (FRET-Expert) asking what linkers they use for fusing GFP at FRET.
• Team Fungal 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 NotI. It showed that all parts are on the correct hight in agarose gel.

Saturday July 21st

• Team Cellulose Binding Domain: made primers for GFP (using [http://partsregistry.org/Part:BBa_I13522 BBa_I13522] as template)adding a C-termial His6tag for easy extraction
  • Jun.Prof Thorsten Seidel answered the mail and said that GFPs are easy to fuse and that they use linkers with four AS:
    • ccg gtc gcc acc Upstream of the GFP
    • gaa agc ggc cgc downstream of the GFP; Which is said to work fine, even with a prolin in it.
  • I decided to also use four linking AS and choose to add a C-terminal GS-Linker ([http://partsregistry.org/Part:BBa_J18920 BBa_J18920]) to the CBDclos which would be four linking AS with the Freiburg-scar (TG).
• Team Bacterial Laccase: Since our PCRs did'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 positiv (more details) results.

Sunday July 22nd

• Team Cellulose Binding Domain: Talked to teammates about the concept and decided to use the exact sequence of [http://partsregistry.org/Part:BBa_J18920 BBa_J18920] (GGCAGC)
  • Checked the CBDs we have for illegal restriction sites - found none, not even NgoMIV or AgeI. So we decided to use a Freiburg-Assembly to build the CBD-GFP fusion protein.

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