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).
• Team Site Directed Mutagenesis: Reviewed all Assembly-Standards and made a list of all illegal restriction-sites:
  • EcoRI, NotI, PstI, SpeI, XbaI (Silver), AgeI, NgoMIV (Freiburg), BamHI, BglII, XhoI (Berkeley)
  • Decided to not care for restriction-sites of the Berkeley-assembly (even if it is a great assembly for protein-fusion), because the used Vector (pSB1C3) already has two XhoI-restriction-sites

Tuesday June 19th

• Team Wiki: While using the lab journal more frequently there came up some questions.
  • How detailed do we plan to write our lab journal 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 lab journal entries
  • we divide our lab journal 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 steady state concentration of the substrates. We have low concentrations and not really study state. We found a transformed equation.
• Team Site Directed Mutagenesis: Imported sequences of most of the used bacterial Laccases into Clonemanager and analysed their restriction-sites:
  • bhal has no illegal restriction-sites
  • ecol has one NgoMIV-restriction-site
  • bpul has one XbaI-Restriction-site and one mutation two AgeI- and two NgoMIV-restriction-sites (Decided to delete the Freiburg-restriction-sites would take too much time)
  • tthl has one PstI-restriction-site and two NgoMIV-restriction-sites (Decided to delete the Freiburg-restriction-sites would take too much time)
  • xccl has two PstI, one AgeI and seven NgoMIV-restriction-sites (Decided not to change the NgoMIV-sites, since to mutate seven would take too much time)

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 lab journal 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 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.

• Team Cultivation & Purification:
  • 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.
  • We prepared the basic media and solutions we will need in the lab.
  • Note: All following BioBricks are cloned into pSB1C3 and therefore cultivated with 20µg/mL chloramphenicol unless otherwise specified!
  • Preparing our first preculture of E.coli KRX containing either the laccases from B.halodurans, X.campestris or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005]. As negative control we used E.coli KRX.

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.

• Team Cultivation & Purification:
  • Today we started our first cultivation of E.coli KRX with laccases from B.halodurans, X.campestris or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005]. As conditions we chosed the ones proposed by [http://www.promega.com/~/media/files/resources/protocols/technical%20bulletins/101/single%20step%20competent%20cells%20protocol.pdf?la=en Promega].

-->Settings: TB medium, with 0,4% glucose and 20µg/mL chloramphenicol added. Induction between OD600 of 1-1,5 with 0,1% rhamnose. We tested 300mL flask with/without baffles with a final volume of 60mL and recorded the growth kinetics and also 1L flask without baffles to produce a higher amount of our protein. The cultivations were stopped in the stationary phase(without baffles after 6 hours and with baffles after 7,5 hours).

• • The cells were harvested and disrupted via sonification in a special buffer for each laccase.

Sunday June 24th

• 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.

55px