Difference between revisions of "Team:Tuebingen/Notebook"
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<div class="contentArea"> | <div class="contentArea"> | ||
| − | <p><b>July 18 | + | <div class="contentRow"> |
| − | + | <h1>July 2016</h1> | |
| + | <p><b>July 18 - July 24</b></p> | ||
| + | Fruitforce: | ||
<p>Recovery of Lactobacillus Johnsonii ATCC33200 from lyophilized stab culture, plated | <p>Recovery of Lactobacillus Johnsonii ATCC33200 from lyophilized stab culture, plated | ||
onto MRS-agar-plate. Liquid Cultures inoculated, Stabs created.</p> | onto MRS-agar-plate. Liquid Cultures inoculated, Stabs created.</p> | ||
| − | <p><b>July 25 | + | <p><b>July 25 - August 01</b></p> |
| − | + | Fruitforce & Clonewars: | |
<p>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.</p> | <p>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.</p> | ||
| + | </div> | ||
| − | + | <div class="contentRow"> | |
| − | <p><b>August 18 | + | <h1>August 2016</h1> |
| − | + | <p><b>August 18 - August 21</b></p> | |
| − | < | + | Fruitforce & Clonewars: |
| − | + | <p>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.</p> | |
| − | < | + | <p>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.</p> |
| − | < | + | Interlab Study: |
| − | < | + | <p>Ligation of all parts into pSB1C3, transformation into DH10beta.</p> |
| − | <p><b>August 22 | + | <p><b>August 22 - August 28</b></p> |
Interlabstudy: | Interlabstudy: | ||
| − | < | + | <p>Performed and completed according to protocol.</p> |
| − | < | + | Fruitforce & Clonewars: |
| − | < | + | <p>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. </p> |
| − | 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. | + | 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.</p> |
| − | + | ||
| − | + | ||
| + | <p><b> August 29 - September 04</b></p> | ||
| + | Fruitforce & Clonewars: | ||
| + | <p>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. </p> | ||
| + | <p>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.</p> | ||
| + | <p>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</p> | ||
| + | </div> | ||
| − | + | <div class="contentRow"> | |
| − | <p><b> | + | <h1>September 2016</h1> |
| − | < | + | <p><b>September 05 - September 11</b></p> |
| − | < | + | <p>Revival of L.Johnsonii from stabs. Glycerol stocks created, stored at -80°C. |
| + | <p>PCR on Streptavidin-Hind, optional RBS T4-holin | ||
| + | <p>Restriction and ligation of ljCas9-parts into pSB1C3 (not successful) | ||
| + | <p>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. </p> | ||
| − | + | <p><b>September 12 - September 18</b></p> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <p><b>12 | + | |
<p>Restriction and ligation of ljCas9-parts, PCR on Ligation (not successful). | <p>Restriction and ligation of ljCas9-parts, PCR on Ligation (not successful). | ||
| − | Inoculation of new L. Johnsonii | + | Inoculation of new L. Johnsonii cultures |
<p>Restriction and Ligation of pNZ124 part1 and pNZ124 part2, transformation into DH10beta (successful). | <p>Restriction and Ligation of pNZ124 part1 and pNZ124 part2, transformation into DH10beta (successful). | ||
| + | <p> Miniprep of BBa_R0011 in pSB1C3; BBa_P0312 in pSB1C3 and BBa_K112806 in pSB1C3 | ||
| + | <p> 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. | ||
| + | <p> Digestion and ligation of BBa_P0312 and ApaI-constPromotor to IL2a.</p> | ||
| − | <p><b>19 | + | <p><b>September 19 - September 25</b></p> |
| − | Retried ligations and transformations of previously unsuccessful parts and ligations (not successful). | + | <p>Retried ligations and transformations of previously unsuccessful parts and ligations (not successful). |
| − | Inoculation of new L. Johnsonii Cultures, measurement of growth rates. | + | <p>Inoculation of new L. Johnsonii Cultures, measurement of growth rates. |
| + | <p> Restriction and ligation of IL2a and BBa_R0011 to IL2. Restriction and ligation of IL2 and Kill1 to C2. | ||
| + | <p>Miniprep and digestion of C1 | ||
| + | <p> transformation and miniprep of BBa_P0440 and BBa_R0040 in pSB1C3 | ||
| + | <p> restriction and digestion of Zinc Finger Parts with several Promoters. restriction analysis showed negative results. | ||
| + | </p> | ||
| − | <p><b> 26 | + | <p><b>September 26 - October 02</b></p> |
| − | Retried ligations and transformations of previously unsuccessful parts and ligations (not successful). | + | <p>Retried ligations and transformations of previously unsuccessful parts and ligations (not successful). |
| + | <p> second digestion and ligation of Kill1 and ApaI-Rha to C1. | ||
| + | <p> restriction of BBa_P0440 and BBa_R0040 shows negative results | ||
| + | <p> transformation and miniprep of BBa_P0440 and BBa_R0040 in pSB1C3 from 2015 | ||
| + | <p> digestion of BBa_P0440 and BBa_R0040 in pSB1C3 from 2015 showed negative results. | ||
| + | <p> PCR of BBa_P0440 and BBa_R0040 in pSB1A2 from 2016 showed the right bands. | ||
| + | <p> PCR amplification of Zinkfinger and Promotors parts. Digestion and ligation of the corresponding PCR-products. | ||
| + | </p> | ||
| + | </div> | ||
| + | <div class="contentRow"> | ||
| + | <h1>October 2016</h1> | ||
| + | <p><b>October 03 - October 09</b></p> | ||
| + | <p>Determined that several parts were incorrectly labeled, which prevented correct assembly. | ||
| + | <p>Inoculation of new L. johnsonii Culture | ||
| + | <p>Genomic DNA extraction of L.johnsonii, PCR on genomic DNA for homology arms (not successful) | ||
| + | <p>PCRs on gBlocks of Fru1, Fru2 with extension primers, adding BbsI-site (successful). Restriction and ligation of fragments. PCR on ligation reaction (not successful). | ||
| + | <p> ligation of the PCR product of BBa_P0440 and BBa_R0040 were further amplified via PCR but did not show the right sized band. | ||
| + | <p> Amplification of Zinc Finger Parts via PCR showed no the ligation of the corresponding parts. | ||
| + | <p>PCR on putative Ketohexokinase, restriction, ligation into pSB1C3 | ||
| + | </p> | ||
| − | + | <p><b>October 10 - October 16</b></p> | |
| − | <p><b> | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
Fruitforce: | Fruitforce: | ||
| − | Restriction of ldh+pSB1C3 (successful) | + | <p>Restriction of ldh+pSB1C3 (successful) |
| − | Restriction of putative eSpCas9-BioBrick (not successful). | + | <p>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). | + | <p>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) | + | <P>PCR on Phosphofructokinase, restriction and ligation into pSB1C3, transformation (successful) |
| − | PCR on Phosphofructokinase, restriction and ligation with ldh+pSB1C3, transformation (successful) | + | <p>PCR on Phosphofructokinase, restriction and ligation with ldh+pSB1C3, transformation (successful) |
| − | Restriction of putative Sacc, ligation with ldh+pSB1C3 and transformation (not successful) | + | <p>Restriction of putative Sacc, ligation with ldh+pSB1C3 and transformation (not successful) |
| − | Restriction of Fru (successful), LDH-Promoter in pSB1C3 (successful). | + | <p>Restriction of Fru (successful), LDH-Promoter in pSB1C3 (successful). |
| − | Ligation of Fru and LDH-Promoter in pSB1C3 (not successful) | + | <p>Ligation of Fru and LDH-Promoter in pSB1C3 (not successful) |
| − | Restriction of LDH-Promoter Phosphofructokinase (successful), Ketohexokinase (successful), pTUM104 (successful), pNZ124+Term.(successful). | + | <p>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). | + | <p>Ligation and transformation of LDH-Promoter Phosphofructokinase with pNZ124+Term (not successful), Ketohexokinase with pTUM104 (not successful). |
| + | </p> | ||
| − | <b><p>17 | + | <b><p>October 17 - October 19</b></p> |
| − | 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). | + | <p>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).</p> |
| + | </div> | ||
</div> | </div> | ||
</html> | </html> | ||
Revision as of 02:33, 20 October 2016
July 2016
July 18 - July 24
Fruitforce:Recovery of Lactobacillus Johnsonii ATCC33200 from lyophilized stab culture, plated onto MRS-agar-plate. Liquid Cultures inoculated, Stabs created.
July 25 - August 01
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 2016
August 18 - August 21
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 - August 28
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.August 29 - September 04
Fruitforce & Clonewars: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
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).
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).
