Team:Bielefeld-Germany/Labjournal/week16

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Contents

Week 16 (08/13 - 08/19/12)

Monday August 13th

  • Team Cultivation & Purification:
    • Today we discussed that we may get activity if we start to purify our laccases. So from now we will purify our products before measuring them. We hope this will bring promising results.
  • Team Site Directed Mutagenesis:
    • Gradient-PCR of Tvel10 55 to 66°C with DMSO (12 Steps) resulted in a lot of product at 55°C and 63°C to 66°C; a little product at 56°-59° and 61°-62°C and no product at 60°C (59°C was the temperature Clonemanager predicted and I used before). Merged the products and cleaned them up.
    • Digested the product with EcoRI, PstI and DpnI.
  • Team Cellulose Binding Domain:
    • We did another PCR on the <partinfo>I15322</partinfo> with the GFP_His-primers with positive result.
    • Plasmid-isolation of two positive clones of CBDclos(T7) followed by digestion with NotI that confirmed that the plasmid is CBDclos(T7). Prepared both for sequencing.
    • Plated three positve CBDcex(T7)-colonies.
    • Restriction of the CBDcex(T7)-PCR-product with EcoRI and PstI to bring it into the pSB1C3-backbone.
    • Also: Restriction of the CBDcex(T7)-PCR-product with NgoMIV and PstI for an assembly.
    • Restriktion of the CBDcex(T7)-PCR-product with AgeI and EcoRI for the assembly.
    • All digestions were done with DpnI to get rid of the templates.
    • Ligation of GFP_His with the pSB1C3-backbone.
    • Ligation of CBDcex(T7) and GFP_His with the pSB1C3-backbone.
    • Transformed all ligations into KRX.

Tuesday August 14th

  • Team Arabidopsis Laccase: Today our cDNA took a bath in ethanol and got cleaned. We are pretty sure our next PCR will be a lot more successful. Check protocols for further information about the cDNA washing via ethanol precipitation.
  • Team Cultivation & Purification:
  • Team Site Directed Mutagenesis:
    • Ligation of tvel (PCR-product) and pSB1C3 and transformation into KRX
    • Gradient-PCRs (55°C bis 72 °C) with xccl-plasmid using xccl-g3633c and xccl-g2247c primer-mixes, respectively, resulted in no product of the right size.

Wednesday August 15th

  • Team Wiki: We set up some more wiki rules. Today´s rules were about citations. We agreed von some standards that will for sure help make the wiki a little prettier. Have a look:
    • Paper: Exampleman M et al. (2002). The example paper. The example journal (Volume): pages.
    • Website: Name of website, URL, Index, date site visited
    • Book: Examplewomen M et al. (1999). The example book. The example publisher (edition).

Next will be a standard for charts.

--> Settings: 1L flasks without baffles, final volume 250mL, autoinduction medium supplemented with 60µg/mL chloramphenicol, 37°C, 120rpm, single determination
  • Team Site Directed Mutagenesis:
    • Colony-PCR of tvel10-colonies resulted in small bands. Quickly explained: tvel10 still had illegal PstI-restrictions-sites. Digestion of tvel10 PCR-product with NotI as well as digestion of RFP-pSB1C3 with NotI

Thursday August 16th

  • Team Cultivation & Purification:
    • Today we harvested the cells from the cultivation on 08/15, disrupted them in equilibration buffer via sonification, centrifugated and purified the supernatant via HisTrap column. The purified samples in 500mM Imidazol were given to the activity test team.
  • Team Site Directed Mutagenesis:
    • Clean-up of tvel10 and pSB1C3 (both cut with NotI); Dephosphorilation of pSB1C3 with SAP and ligation. Transformed into KRX and plated on select-agar.

Friday August 17th

Saturday August 18th

  • Team bacterial laccases: Today we performed a ligation from the digestions we have done before. The ligation included of course the T7 promotor, the backbone pSB1C3 and the individual laccase genes. Ligation was performed with T4 ligase for 20 minutes at room temperature and stopped through a five minute incubation at 70°C.

Sunday August 19th

  • Team Site Directed Mutagenesis:
    • Colony-PCR of tvel10-colonies with three positive result.
    • Plated those three on select-agar for plasmid-isolation.
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