Team:Tuebingen/Notebook

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July 18.07.2016-24.07.2016

Fruitforce:

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

July 25.07.2016-01.08.2016:

Fruitforce & Clonewars:

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 18.08.2016-21.08.2016

Fruitforce & Clonewars:

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 22.08.2016-28.08.2016

Interlabstudy:

Performed and completed according to protocol. Fruitforce & Clonewars:

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.

29.08.2016-04.09.2016

Fruitforce & Clonewars:

Ligation of pNZ124 part1 with pNZ124 part2 from gBlocks .

05.09.2016-11.09.2016

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)

12.09.2016-18.09.2016

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).

19.09.2016-25.09.2016:

Retried ligations and transformations of previously unsuccessful parts and ligations (not successful). Inoculation of new L. Johnsonii Cultures, measurement of growth rates.

26.09.2016-02.10.2016

Retried ligations and transformations of previously unsuccessful parts and ligations (not successful).

03.10.2016-09.10.2016

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). PCR on putative Ketohexokinase, restriction, ligation into pSB1C3

10.10.2016-16.10.2016

Fruitforce: 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).

17.10.2016-19.10.2016

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).