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 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 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).