Team:Bielefeld-Germany/Labjournal/week8

Week 8 (06/18 - 06/24/12)

Monday June 18th

• Team Bacterial Laccases: 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 the Tth laccase, not for CotA (B. pumilus).

Tuesday June 19th

• Team Wiki: While using the labjournal more frequently there came up some questions.
  • How detailed do we plan to write our labjournal entries?
  • Do we want to write in keywords or explain everything in full sentences?
  • Do we want to note every little detail about every successful or unsuccessful experiment or just the main important aspect?

We discussed, sighted some former iGEM team wikis and decided:

• • each team is responsible for their own labjournal entries
  • we divide our labjournal in weeks and days to prevent it from looking too chaotic.
  • 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.

Wednesday June 20th

• Team Modeling: Finding out, that the "normal" Michaelis-Menten kinetic isn't the right kinetic to model our situation, because therefor you need a high and stady state çoncentration of the subtrates. We have low concentrations and not really stady state. We found a transformed equation.

Thursday June 21st

• Team Activity Tests and Team Immobilization: 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 labjournal Team Immobilization).

• Team Bacterial Laccases: Plasmid isolation and control restriction with NotI on plasmids with Tth laccase and luckily this time the bands were where they should be. Again and again..transformation of ligation with CotA laccase in pSB1C3 backbone..all fingers are crossed that this time we have colonies with th correct plasmid.

Friday June 22nd

• Team Cultivation/Purification: Starting our first cultivation experiments with our first Biobricks. Transformed cells are cultivated over night for our first preculture. After finishing our work in the laboratory we decided to collect some information for the best cultivation conditions. This night we find an interesting report of the Deutsche Bundesstiftung Umwelt (DBU) with interesting fact about different Laccases. With this Report we decided to screen different forms of th Erlenmeyer flasks.

• Team Bacterial Laccases: 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.

Saturday June 23rd

• Team Bacterial Laccases:
  • 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.
  • Colony PCRs on the transformations with plasmid with CotA laccase from Bacillus pumilus and plating positive colonies.

Sunday June 24th

• Team Activity Test: Today we received the first laccases from Team Cultivation. We have been waiting for this moment full of anticipation. We tested them under our standard conditions meaning an addition of 0,1 mM ABTS. The laccases were already waiting for us in the appropriate and optimal buffer system (according to the literature, please check Team Cultivations labjournal). Tecan measured each sample for 10 minutes and told us about the according OD values afterwards. Unfortunately no change in the OD values was observed by Tecan and us. We felt like we could not take the laziness of our laccases. We thought and discussed what the reason could be for them to be inactive.
  • Is there something wrong with the transformed construct, maybe a mutation?
  • Was the laccase synthesized but is inactive for some reason?
  • Was the laccase produced but not in a high enough amount so that we can´t detect its activity in the first place?

Another thought was that there might be copper missing. Since laccase is a copper-enzyme it might be necessary to add copper to the medium whenever it is produced in such high amounts. It´s late, so we will do that tomorrow.

• Team Bacterial Laccases: We isolated plasmids and did control digests with NotI. We finally had a positive restriction digest for the CotA (B. pumilus) plasmid. So we prepared this plasmids and the plasmids with the Tth laccase which we isolated some days before for sequencing.

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