Team:Tuebingen/Notebook

July 2016

July 18 - July 24

  • Recovery of Lactobacillus Johnsonii ATCC33200 from lyophilized stab culture, plated onto MRS-agar-plate. Liquid Cultures inoculated, Stabs created.

July 25 - August 01

  • Ligation of putative gBlocks ApaI-pRha, ApaI-constPromotor,Apa-Bam-Promotor-C7C7bind-RBS, Apa-Bam-Promotor-K230Rbind-RBS,Apa-Bam-Promotor-ZFNLbind-RBS, RNA promotor-Idh, Terminator-Bam-Hind, ZF ZFN-L, ZF K230R and ZF-C7C7 into pSB1C3, transformation into DH10beta.

August 2016

August 18 - August 21

  • PCR on minipreps of ApaI-pRha, ApaI-constPromotor, Apa-Bam-Promotor-C7C7bind-RBS, Apa-Bam-Promotor-K230Rbind-RBS, Apa-Bam-Promotor-ZFNLbind-RBS, RNA promotor-Idh, Terminator-Bam-Hind, ZF ZFN-L, ZF K230R and ZF-C7C7 in pSB1C3.
  • Ligation of putative gBlocks sgRNA scaffold with promotor, FokI-part1, FokI-part2, Ij Cas part1, Ij Cas part2, Ij Cas part3, Saccharose Transporter part 1, Saccharose Transporter part 2, RBS-HSV-Tk, RBS-PezT, Phosphofructokinase and Ketohexokinase for Yeast into pSB1C3, transformation into DH10beta.
  • Interlab Study: Ligation of all parts into pSB1C3, transformation into DH10beta.

August 22 - August 28

  • Interlabstudy: Performed and completed according to protocol.
  • Restriction of putative Fru1, Fru2, ligation into pSB1C3 and transformation. PCR of ApaI-pRha; ApaI-constPromotor; Apa-Bam-Promotor-C7C7bind-RBS; Apa-Bam-Promotor-K230Rbind-RBS; Apa-Bam-Promotor-ZFNLbind-RBS; RNA promotor-Idh; Terminator-Bam-Hind; ZF ZFN-L; ZF K230R; ZF-C7C7; Streptavidin-Hind; RBS T4-holin; sgRNA scaffold with promotor; RBS-HSV-Tk; RBS-PezT; Phosphofructokinase; Ketohexokinase; from g-Blocks.
  • Ligation of Streptavidin-Hind; Fokl-linked part 1; Fokl-linked part 2; Ij Cas part1; Ij Cas part2; Ij Cas part3; Saccharose Transporter part 1; Saccharose Transporter part 2; Fructosetransporter part 1; Fructosetransporter part 2; from PCR-Products into pSB1A2.

August 29 - September 04

  • Ligation of pNZ124 part1 with pNZ124 part2 from gBlocks and transformation into cells (not successful); Restriction of RBS-HSV-Tk; ZF K230R;Saccharose Transporter part 2; Phosphofructokinase from pSB1C3 Plasmid. Restriction of Fructosetransporter part 2 from gBlock.
  • Miniprep of ApaI-pRha; ApaI-constPromotor; Apa-Bam-Promotor-C7C7bind-RBS; Apa-Bam-Promotor-K230Rbind-RBS; Apa-Bam-Promotor-ZFNLbind-RBS; RNA promotor-Idh; Terminator-Bam-Hind; ZF ZFN-L; ZF K230R; ZF-C7C7; sgRNA scaffold with promotor; Fokl-linked part 1; Fokl-linked part 2; Saccharose Transporter part 1; Saccharose Transporter part 2; Phosphofructokinase; Ketohexokinase for yeast; integrated in pSB1C3 plasmid.
  • Restriction of RBS T4-holin; Ij Cas part1; Ij Cas part2; Ij Cas part3; Fokl-linked part 1; Fokl-linked part 2; Saccharose Transporter part 1; Saccharose Transporter part 2; Phosphofructokinase; sgRNA scaffold with promotor and RNA promotor-Idh integrated in pSB1C3 plasmid

September 2016

September 05 - September 11

  • Revival of L.Johnsonii from stabs. Glycerol stocks created, stored at -80°C.
  • PCR on Streptavidin-Hind, optional RBS T4-holin
  • Restriction and ligation of ljCas9-parts into pSB1C3 (not successful)
  • Restriction of Terminator-Bam-Hind from pSB1C3. Transformation and inoculation of primary culture with BBa_R0011 in pSB1C3; BBa_P0312 in pSB1C3 and BBa_K112806 in pSB1C3.

September 12 - September 18

  • Restriction and ligation of ljCas9-parts, PCR on Ligation (not successful).
  • Inoculation of new L. Johnsonii cultures
  • Restriction and Ligation of pNZ124 part1 and pNZ124 part2, transformation into DH10beta (successful).
  • Miniprep of BBa_R0011 in pSB1C3; BBa_P0312 in pSB1C3 and BBa_K112806 in pSB1C3
  • Restriction of RBS-PezT. Ligation of RBS-PezT with Terminator-Bam-Hind to Kill1. Transformation and miniprep of Kill1. Digestion and ligation of Kill1 and ApaI-Rha to the construct C1.
  • Digestion and ligation of BBa_P0312 and ApaI-constPromotor to IL2a.

    • September 19 - September 25

    • Retried ligations and transformations of previously unsuccessful parts and ligations (not successful).
    • Inoculation of new L. Johnsonii Cultures, measurement of growth rates.
    • Restriction and ligation of IL2a and BBa_R0011 to IL2. Restriction and ligation of IL2 and Kill1 to C2.
    • Miniprep and digestion of C1
    • transformation and miniprep of BBa_P0440 and BBa_R0040 in pSB1C3
    • restriction and digestion of Zinc Finger Parts with several Promoters. restriction analysis showed negative results.

    September 26 - October 02

    • Retried ligations and transformations of previously unsuccessful parts and ligations (not successful).
    • second digestion and ligation of Kill1 and ApaI-Rha to C1.
    • restriction of BBa_P0440 and BBa_R0040 shows negative results
    • transformation and miniprep of BBa_P0440 and BBa_R0040 in pSB1C3 from 2015
    • digestion of BBa_P0440 and BBa_R0040 in pSB1C3 from 2015 showed negative results.
    • PCR of BBa_P0440 and BBa_R0040 in pSB1A2 from 2016 showed the right bands.
    • PCR amplification of Zinkfinger and Promotors parts. Digestion and ligation of the corresponding PCR-products.

    October 2016

    October 03 - October 09

    • Determined that several parts were incorrectly labeled, which prevented correct assembly.
    • Inoculation of new L. johnsonii Culture
    • Genomic DNA extraction of L.johnsonii, PCR on genomic DNA for homology arms (not successful)
    • PCRs on gBlocks of Fru1, Fru2 with extension primers, adding BbsI-site (successful). Restriction and ligation of fragments. PCR on ligation reaction (not successful).
    • ligation of the PCR product of BBa_P0440 and BBa_R0040 were further amplified via PCR but did not show the right sized band.>
    • Amplification of Zinc Finger Parts via PCR showed no the ligation of the corresponding parts.
    • PCR on putative Ketohexokinase, restriction, ligation into pSB1C3

    October 10 - October 16

    • Restriction of ldh+pSB1C3 (successful)
    • Restriction of putative eSpCas9-BioBrick (not successful).
    • PCRs on gBlocks of Fru1, Fru2 with extension primers, adding BbsI-site. Restriction and ligation of fragments. PCR on ligation reaction, gel purification. Restriction and ligation into pSB1C3, transformation into DH5a (all successful).
    • PCR on Phosphofructokinase, restriction and ligation into pSB1C3, transformation (successful)
    • PCR on Phosphofructokinase, restriction and ligation with ldh+pSB1C3, transformation (successful)
    • Restriction of putative Sacc, ligation with ldh+pSB1C3 and transformation (not successful)
    • Restriction of Fru (successful), LDH-Promoter in pSB1C3 (successful).
    • Ligation of Fru and LDH-Promoter in pSB1C3 (not successful)
    • Restriction of LDH-Promoter Phosphofructokinase (successful), Ketohexokinase (successful), pTUM104 (successful), pNZ124+Term.(successful).
    • Ligation and transformation of LDH-Promoter Phosphofructokinase with pNZ124+Term (not successful), Ketohexokinase with pTUM104 (not successful).

    October 17 - October 19

    • Test restrictions of Kill1, ZF ZFN-L, ZF K230R, ZF-C7C7, PezT, Term. Bam-Hind, ApaI const. Promoter, Ketohexokinase for Yeast, Phosphofructokinase, LDH-Promoter Phosphofructokinase, LDH-Promoter and D2 (all successful).