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 Fungal Laccases: Digestion 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.pumilus 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.
• After our laccases seemed not to get produced we decided to try to express the laccases with constitutive promoters. Therefore we designed primers with the sequences of different promoters from the [Enderson promoter family]. We chose the promoter sequences of the parts BBa_J23103, BBa_J23110 and BBa_J23117 and for all three promoters the RBS BBa_B0034. The primers were designed that the FW and the RV primers ligate to a short oligonucleotide with overhanging restriction site ends for EcoRI and SpeI. So we don’t have to cut the ligated primers, because the sites should appear with the correct annealing. The goal was to clone the different promoters, the PCR product with the different laccase genes with HIS-Tag with prefix and suffix in pSB1C3 in one ligation step.

• Additionally we want to produce the constructs with a new t7 promoter. After our laccases were not expressed we now think that maybe the RBS BBa_B0034, which we changed from originally 5'aAagaggagaaa3' to 5'aGagaggagaaa3' is not or to less recognized from ribosomes in the cells. This is the case, because for designing our T7 promoter overhanging ends for our primers we chose the same sequence as the Bielefeld Team before for example in their part BBa_K525710. But another sequence for a T7 promoter, which is described in the Parts Registry was the sequence of the part BBa_I719005. So we changed the sequence they used in their part BBa_K525710 from 5'taatacgactcactatagggaAagaggagaaaa 3' to 5’taatacgactcactatagggaGagaggagaaaa 3’ and used this sequence in our primers so the T7 sequence is the one from part BBa_I719005.

We have a primer pair from last year with the T7 promoter sequence with RBS BBa_B0034. The primers can be annealed to an oligonucleotide. After boiling the primers and cooling down there should be an oligonucleotide with an EcoRI and a SpeI restriction site. We now want to assemble the promoter the laccase genes and the pSB1C3 vector in one step. The pSB1C3 backbone was digested with EcoRI and PstI, the laccases with XbaI and PstI, and the promoter has the restriction sites SpeI and EcoRI.

• Team Fungal Laccases: Purification of Tv35 PCR product and digestion for cloning in pSB1C3 backbone.

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
• Team Bacterial Laccases: With the new T7 promoter we started new assemblies. We digested our laccase PCR products for suffix insertion with XbaI and PstI, boiled and cooled down the joined T7 primer pairs (should have EcoRI and SpeI overhangs after annealing) and digested pSB1C3 backbone with EcoRI and PstI.

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
• Team Bacterial Laccases: Transformation of the ligated assemblies (E.coli, B. pumi, B. halo, Xcc, Tth laccase PCR products) from the day before.
• Team Fungal Laccases: Ligation of Tv5 and Tv35 in pSB1C3.

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 fermantation group in KRX an plated them on Kanamycin-selectionagar.
• Team Fungal Laccases: Transformation of Tv5 and Tv35 in pSB1C3 backbone.
• Team Bacterial Laccases: The assembly from the last two days seem not work. All colonies we have are red due to the original plasmid with RFP we isolated the backbone from.

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
• Team Bacterial Laccases: We did the transformation of the ligations from August 1st again.

Sunday August 5th

• Team Site Directed Mutagenesis: Plasmid isolations 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 positive, 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
  • Plasmid isolations of p570
  • PCR of CBDcex (417 bp) and CBDclos (369 bp)
    • Test gel electrophoresis: Got the bands that should be there!
    • PCR Clean up (no Gelelectrophoresis)
  • Restriction of PCR-products and pSB1C3_RFP with XbaI and PstI.

• Team Bacterial Laccases: Did the restriction and the ligation of E.coli P/S and E. coli HIS in pSB1C3.

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