Team:Bielefeld-Germany/Labjournal/week14

Week 14 (07/30 - 08/05/12)

Monday July 30th

• Team Site Directed Mutagenesis: Preparations for plasmid-isolations of B.pumi_2883_IV (I-IV); X.camp_3633 (I-IV); X.camp._2247_2 (I-IV) and B.pumi_2883_III (I-IV)
• Team Cellulose Binding Domain: Redesigned the primer-sequences another time giving the CBDs a few AS more in than the Protein-BLAST said. CBDcex: 4 AS N-terminal, 2 C-terminal. CBDclos: 2 N-terminal (starting with a natural ATG) and 2 AS C-terminal.
  • Prepared BBa_392014 for Sequencing
• Team Bacterial Laccases: Restriction of Tv5 laccase PCR product with Prefix and Suffix ends for cloning in pSB1C3 and restriction of laccase with the overhangs for cloning in shuttle vector. And we did the PCR on Tv35 with both primer pairs again. This time we lowered the annealing temperatures and got products with both primer pairs.

Tuesday July 31st

• Team Site Directed Mutagenesis: Picked four colonies per dish T.vers._243 & T.vers._1161 and plated them for plasmid-isolation.
  • Plasmid-isolation of B.pumi III(III)+IV(IV) with SDM-Mix 2317 X.camp'
  • Test-restriction 700ng in 10 µL with PstI

Results of cutting if mutation failed:

The B.pumilus site directed mutation at 2317 is not a illegal restriction site, so it can not be checked via restriction, but only through sequencing.

• Picked a few more Colonies of X.camp for plasmid-isolation

• Team Bacterial Laccases:

• Ligation of Tv5 laccase with pSB1C3 backbone and purification of Tv35 laccase PCR products.
• We designed primers with the sequences of different promoters from the [Enderson promotor family]. We chose the sequences of the parts BBa_J23103, BBa_J23110 and BBa_J23117.

Wednesday August 1st

• Team Site Directed Mutagenesis: Plasmid isolation of T.vers_243 (I-IV) & T.v._1161 (I-IV) X.camp_2K&3K (V-VI)
  • Test-restriction with PstI / SpeI & Anaylsis via Gelelectrophoresis show bands as they should for T.vers_243 II+III; and X.camp._2K_2 V
  • Made X.camp._2K_2 V and the final B.pumilusplasmids ready for sequencing

Thursday August 2nd

• Team Wiki: This morning we met for taking a new picture of our group and individual pictures of everyone. Check out our beautiful team members here.
• Team Site Directed Mutagenesis: SDM PCR of T.vers243 II+III with 1161 Primer mix and X.camp._2K_2 V with 3633

Friday August 3rd

• Team Site Directed Mutagenesis: Transformed T.vers_243_III_1161 and X.camp._2K_2 V_3633 in XL1 Blue and plated it on Ampicillin- and CM-selection-agar.
• Team Cellulose Binding Domain: Transformed the plasmids p714_cex and 570_clos we got from the fermantationgroup in KRX an plated them on Kanamycin-selectionagar.

Saturday August 4th

• Team Site Directed Mutagenesis: Plated four isolated colonies from T.vers_243_III_1161 and X.camp._2K_2 V_3633 per petri dish on seperated selection-agar-dishes

• Team Cellulose Binding Domain: Since there were only a few colonies on the p570 selection-agar-dish, I picked one, resuspended it in 100 µL destilled water and plated it on an new Kanamycin-selection-agar-dish
  • plasmid-isolation of p714

Sunday August 5th

• Team Site Directed Mutagenesis: Plasmidisolations of X.camp.2K_2_3633 (I-IV) and T.vers_243_III_1161 (I-IV)
  • Test-Restriktion of X.camp.2K_2_3633 (I-IV) and T.vers_243_III_1161 (I-IV) X.camp III + IV good; T.vers, no positiv, since the 1150 band should at least rise to 1550
  • Picked two additional colonies of e.coli_2307 and plated them on CM
  • Prepared X.camp.2K_2_3633 III + IV for sequencing
• Team Cellulose Binding Domain: Transformation of BBa_I13522 + pSB1A2 in KRX and plating it on AMP-Selection-Agar
  • Plasmidisolations of p570
  • PCR of CBDcex (417 bp) and CBDclos (369 bp)
    • Test-Gelelectrophoresis: Got the bands that should be there!
    • PCR Clean up (no Gelelectrophoresis)
  • Restriktion of PCR-products and pSB1C3_RFP with XbaI and PstI.

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