Team:Bielefeld-Germany/Labjournal/week7

Week 7 (06/11 - 06/17/12)

weekly seminar

• Agatha and Saskia will perform a mRNA isolation from Arabidopsis thaliana.
• Everything is in progress. Check.

Monday June 11th

• Team Cloning of Bacterial Laccases:
  • Prepared plasmids for sequencing. We sent another isolated plasmid with'ecol(T7)_His. Also tthl(t7)_His, bahl(T7)_His and bpul(T7)_His plasmids were ready for sequencing.
• Team Fungal and Plant Laccases:
  • We looked for the sequences of the laccase of Trametes versicollor in different databases. By searching the specific sequences we realized, that we have to pay special attention to the strains the sequnces came from. During our search we cannot find any published strain in the DMSZ or other straincollections. So we decided to write e-mails to different working groups all over the world to get the published strains.
• Team Immobilization:
  • Today, we took our first step: we started searching for publications which describe different methods for the immobilization of laccases, especially those from Trametes versicolor in order to figure out the best method that suits our laccases. Some of the papers are listed below:
    • [http://www.ncbi.nlm.nih.gov/pubmed/22398306 Recent developments and applications of immobilized laccase.]
    • [http://www.hindawi.com/journals/er/2011/725172/ Enzyme-Catalyzed Oxidation of 17β-Estradiol Using Immobilized Laccase from Trametes versicolor]
    • [http://www.ncbi.nlm.nih.gov/pubmed/17917725 Reactive blue 19 decolouration by laccase immobilized on silica beads]
    • [http://www.ncbi.nlm.nih.gov/pubmed/16988786 Alginate/carbon composite beads for laccase and glucose oxidase encapsulation: application in biofuel cell technology.]

Tuesday June 12th

• Team Student Academy:
  • The whole experiment was tested by another team member to plan the course.

Wednesday June 13th

• Team Cloning of Bacterial Laccases: Since the GC amount of the S. griseus and S. lavendulae laccases are high we used betain to solve the PCR problem. Addition of betain did not change anything on the results, we still didn't got our laccase DNA.

• Team Modeling: Programming our first differential equation and finding the ODE15s function witch solves these equations.

Thursday June 14th

• Team Cloning of Bacterial Laccases:
  • Because our PCRs have not worked well we thought it may depends on the primer annealing temperature so we did gradient PCR with the same conditions as before (PCR June 4th). But this also showed no result. Because we made Coloyn PCRs from the arrived DSMZ reaction tubes our next idea was to cultivate the bacteria in media and isolate genomic DNA.

• Team Activity Tests:
  • Since our Tecan microplate reader is not able to actively cool down to 4 °C we got the chance to meet the photometer Carry. Check "protocols" for further information about her. We used the same set up with 100 mM natrium acetate buffer, 0,1 U T. versicolor laccase and 0,1 mM ABTS as before but now measured at 4°C. Our team is planning to visit a municipal sewage plant for getting some insights into the water conditions there, so we will for sure test other temperatures after having more information. Let´s hope the water there is a little warmer since laccase does not seem to be totally satisfied at 4°C. I would not either.

Friday June 15th

• Team Cloning of Bacterial Laccases:
  • Sequencing of the pSB1C3 plasmid with bhal(T7)_His was ok. In conflict to our reference sequence there was a point mutation in the DNA sequence but this mutation doesn’t lead to another amino acid. So..next BioBrick (<partinfo>BBa_K863020</partinfo>) is ready to use!
  • The sequenced plasmid bpul(T7)_His showed again the same mutation in the laccase ORF compared to the reference sequence. We concluded that probably the PCR amplification caused the point mutation. So we did the digest of bpul(T7)_His PCR products from a new PCR, ligated it in pSB1C3 backbone and transformed it in competent KRX cells. Additionally we did the digest tthl(T7)_His and the ligation in pSB1C3 backbone again.
• Team Fungal and Plant Laccases:

The whole week we were looking for different sources of the laccase sequences and the corresponding strain, we find an interessting study of the Federal Environmental Agency (Umwelt-Bundes-Amt Germany). In this study they analysed the oppurtunities to synthesis different chemical compounds with the aid of different laccase of Tramtetes versicolor. In this document we found a working group from the Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis at Ernst-Moritz-Arndt-University in Greifswald (Germany) who have isolated four sequences of different laccases of Trametes versicolor and the sequence of one Pycnoporus cinnabarinus. We have send a request for the plasmids containing cDNA sequences of five different laccases from Trametes versicolor and Pycnoporus cinnabarinus .

Saturday June 16th

Sunday June 17th

55px