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Team:Bielefeld-Germany/Labjournal/week7
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* '''Team Bacterial Laccases''': | * '''Team Bacterial Laccases''': | ||
** Sequencing of the pSB1C3 plasmid with the CotA laccase from ''B. halodurans'' 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 ready to use! | ** Sequencing of the pSB1C3 plasmid with the CotA laccase from ''B. halodurans'' 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 ready to use! | ||
| - | ** The sequenced product of the plasmid with the ''B. pumilus'' laccase 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 CotA (''B. pumilus'') PCR products from a PCR we did before, ligated it in pSB1C3 backbone and transformed it in competent KRX cells. | + | ** The sequenced product of the plasmid with the ''B. pumilus'' laccase 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 CotA (''B. pumilus'') PCR products from a PCR we did before, ligated it in pSB1C3 backbone and transformed it in competent KRX cells. Additionally we did the digest of the T.th laccase and the ligation in pSB1C3 backbone again. |
===Saturday June 16th=== | ===Saturday June 16th=== | ||
Revision as of 01:44, 14 September 2012
Contents |
Week 7 (06/11 - 06/17/12)
Monday June 11th
- Team Bacterial Laccases: Prepared plasmids for sequencing. We sent another isolated plasmid with the E. coli CueO laccase. Also the Tth- laccase, CotA (B. halodurans and CotA (B.pumilus) plasmids were ready for sequencing.
Tuesday June 12th
Wednesday June 13th
- Team Modeling: Programming our first differential equation and finding the ODE15s function witch solves these equations.
- Team 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 of the result, we still didn't got our laccase DNA.
Thursday June 14th
- 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.
- Team Bacterial laccase: 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 conductions as before (PCR June 4th). But this also showed no result. Our next idea was to let the bacteria grow in media and isolate genomic DNA.
Friday June 15th
- Team Bacterial Laccases:
- Sequencing of the pSB1C3 plasmid with the CotA laccase from B. halodurans 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 ready to use!
- The sequenced product of the plasmid with the B. pumilus laccase 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 CotA (B. pumilus) PCR products from a PCR we did before, ligated it in pSB1C3 backbone and transformed it in competent KRX cells. Additionally we did the digest of the T.th laccase and the ligation in pSB1C3 backbone again.
Saturday June 16th
Sunday June 17th
Sunday
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