iGEM 2016 — Team Tuebingen

[![](https://static.igem.org/mediawiki/2016/8/8b/HQ_page_logo.jpg)](https://2016.igem.org)

# Team:Tuebingen/Notebook

![](https://static.igem.org/mediawiki/2016/1/16/T--Tuebingen--Logo_iGem.png)

* Team
  + [Home](https://2016.igem.org/Team%3ATuebingen)+ [The Team](https://2016.igem.org/Team%3ATuebingen/Team)
* Fruitforce
  + [Introduction](https://2016.igem.org/Team%3ATuebingen/FruitSummary)
    #+ [L. johnsonii](https://2016.igem.org/Team%3ATuebingen/L_johnsonii)+ [Project design](https://2016.igem.org/Team%3ATuebingen/FruitDesign)+ [Model](https://2016.igem.org/Team%3ATuebingen/Model)+ [Results](https://2016.igem.org/Team%3ATuebingen/Results)
* Side Projects
  + [Clone Wars](https://2016.igem.org/Team%3ATuebingen/CloneWars)+ [Death Star](https://2016.igem.org/Team%3ATuebingen/DeathStar)+ [Interlab Study](https://2016.igem.org/Team%3ATuebingen/Measurement)
* Lab
  + [Parts](https://2016.igem.org/Team%3ATuebingen/Parts)+ [Notebook](https://2016.igem.org/Team%3ATuebingen/Notebook)+ [Safety](https://2016.igem.org/Team%3ATuebingen/Safety)+ [Methods](https://2016.igem.org/Team%3ATuebingen/Methods)
* HP
  + [Summary](https://2016.igem.org/Team%3ATuebingen/HP/Silver)+ [Internship](https://2016.igem.org/Team%3ATuebingen/HP/Gold)+ [Symposium Marburg](https://2016.igem.org/Team%3ATuebingen/HP/Marburg1)+ [iGEM Marburg](https://2016.igem.org/Team%3ATuebingen/HP/Marburg2)+ [EE16 Paris](https://2016.igem.org/Team%3ATuebingen/HP/Paris)+ [Science Slam](https://2016.igem.org/Team%3ATuebingen/HP/Constance)+ [Science Pub Tue](https://2016.igem.org/Team%3ATuebingen/HP/SciencePub)+ [Symposium Tue](https://2016.igem.org/Team%3ATuebingen/HP/Symposium)
* Attributions
  + [Collaborations](https://2016.igem.org/Team%3ATuebingen/Collaborations)+ [Sponsoring](https://2016.igem.org/Team%3ATuebingen/Sponsoring)+ [Acknowledgements](https://2016.igem.org/Team%3ATuebingen/Attributions)+ [References](https://2016.igem.org/Team%3ATuebingen/References)

# 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).

[![](https://static.igem.org/mediawiki/2016/b/bf/HQ_menu_logo.jpg)](https://2016.igem.org)