Difference between revisions of "Team:Tuebingen/Notebook"

 
(One intermediate revision by the same user not shown)
Line 11: Line 11:
 
<h1>July 2016</h1>
 
<h1>July 2016</h1>
 
<p><b>July 18 - July 24</b></p>
 
<p><b>July 18 - July 24</b></p>
−
Fruitforce:
+
<ul>
−
<p>Recovery of Lactobacillus Johnsonii ATCC33200 from lyophilized stab culture, plated  
+
<li>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.</li>
 +
</ul>
  
 
<p><b>July 25 - August 01</b></p>
 
<p><b>July 25 - August 01</b></p>
−
Fruitforce & Clonewars:
+
<ul>
−
<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>
+
<li>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.</li>
 +
</ul>
 
</div>
 
</div>
 
   
 
   
Line 23: Line 25:
 
<h1>August 2016</h1>  
 
<h1>August 2016</h1>  
 
<p><b>August 18 - August 21</b></p>
 
<p><b>August 18 - August 21</b></p>
−
Fruitforce & Clonewars:
+
<ul>
−
<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>
+
<li>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.</li>
−
<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>
+
<li>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.</li>
−
Interlab Study:
+
<li>Interlab Study: Ligation of all parts into pSB1C3, transformation into DH10beta.</li>
−
<p>Ligation of all parts into pSB1C3, transformation into DH10beta.</p>
+
</ul>
  
 
<p><b>August 22 - August 28</b></p>
 
<p><b>August 22 - August 28</b></p>
−
Interlabstudy:
+
<ul>
−
<p>Performed and completed according to protocol.</p>
+
<li>Interlabstudy: Performed and completed according to protocol.</li>
−
Fruitforce & Clonewars:
+
<li>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. </li>
−
<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>
+
<li>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.</li>
−
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>
+
</ul>
 
   
 
   
 
<p><b> August 29 - September 04</b></p>
 
<p><b> August 29 - September 04</b></p>
−
Fruitforce & Clonewars:
+
<ul>
−
<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>
+
<li>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. </li>
−
<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>
+
<li>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.</li>
−
<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>
+
<li>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</li>
 
</div>
 
</div>
  
Line 46: Line 48:
 
<h1>September 2016</h1>
 
<h1>September 2016</h1>
 
<p><b>September 05 - September 11</b></p>
 
<p><b>September 05 - September 11</b></p>
−
<p>Revival of L.Johnsonii from stabs. Glycerol stocks created, stored at -80°C.
+
<ul>
−
<p>PCR on Streptavidin-Hind, optional RBS T4-holin
+
<li>Revival of L.Johnsonii from stabs. Glycerol stocks created, stored at -80°C.</li>
−
<p>Restriction and ligation of ljCas9-parts into pSB1C3 (not successful)
+
<li>PCR on Streptavidin-Hind, optional RBS T4-holin</li>
−
<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>   
+
<li>Restriction and ligation of ljCas9-parts into pSB1C3 (not successful)</li>
 +
<li>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. </li>   
 +
</ul>
  
 
<p><b>September 12 - September 18</b></p>
 
<p><b>September 12 - September 18</b></p>
−
<p>Restriction and ligation of ljCas9-parts, PCR on Ligation (not successful).
+
<ul>
−
Inoculation of new L. Johnsonii cultures
+
<li>Restriction and ligation of ljCas9-parts, PCR on Ligation (not successful).</li>
−
<p>Restriction and Ligation of pNZ124 part1 and pNZ124 part2, transformation into DH10beta (successful).
+
<li>Inoculation of new L. Johnsonii cultures</li>
−
<p> Miniprep of BBa_R0011 in pSB1C3; BBa_P0312 in pSB1C3 and BBa_K112806 in pSB1C3  
+
<li>Restriction and Ligation of pNZ124 part1 and pNZ124 part2, transformation into DH10beta (successful).</li>
−
<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.  
+
<li> Miniprep of BBa_R0011 in pSB1C3; BBa_P0312 in pSB1C3 and BBa_K112806 in pSB1C3 </li>
−
<p> Digestion and ligation of BBa_P0312 and ApaI-constPromotor to IL2a.</p>  
+
<li> 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. </li>
 +
<li> Digestion and ligation of BBa_P0312 and ApaI-constPromotor to IL2a.</li>
 +
</ul> 
  
 
<p><b>September 19 - September 25</b></p>
 
<p><b>September 19 - September 25</b></p>
−
<p>Retried ligations and transformations of previously unsuccessful parts and ligations (not successful).
+
<ul>
−
<p>Inoculation of new L. Johnsonii Cultures, measurement of growth rates.
+
<li>Retried ligations and transformations of previously unsuccessful parts and ligations (not successful).</li>
−
<p> Restriction and ligation of IL2a and BBa_R0011 to IL2. Restriction and ligation of IL2 and Kill1 to C2.  
+
<li>Inoculation of new L. Johnsonii Cultures, measurement of growth rates.</li>
−
<p>Miniprep and digestion of C1   
+
<li> Restriction and ligation of IL2a and BBa_R0011 to IL2. Restriction and ligation of IL2 and Kill1 to C2. </li>
−
<p> transformation and miniprep of BBa_P0440 and BBa_R0040 in pSB1C3
+
<li>Miniprep and digestion of C1  </li>
−
<p> restriction and digestion of Zinc Finger Parts with several Promoters. restriction analysis showed negative results.  
+
<li> transformation and miniprep of BBa_P0440 and BBa_R0040 in pSB1C3</li>
−
</p>
+
<li> restriction and digestion of Zinc Finger Parts with several Promoters. restriction analysis showed negative results. </li>
 +
</ul>
  
 
<p><b>September 26 - October 02</b></p>
 
<p><b>September 26 - October 02</b></p>
−
<p>Retried ligations and transformations of previously unsuccessful parts and ligations (not successful).  
+
<ul>
−
<p> second digestion and ligation of Kill1 and ApaI-Rha to C1.
+
<li>Retried ligations and transformations of previously unsuccessful parts and ligations (not successful).</li>
−
<p> restriction of BBa_P0440 and BBa_R0040 shows negative results  
+
<li> second digestion and ligation of Kill1 and ApaI-Rha to C1.</li>
−
<p> transformation and miniprep of BBa_P0440 and BBa_R0040 in pSB1C3 from 2015
+
<li> restriction of BBa_P0440 and BBa_R0040 shows negative results </li>
−
<p> digestion of BBa_P0440 and BBa_R0040 in pSB1C3 from 2015 showed negative results.  
+
<li> transformation and miniprep of BBa_P0440 and BBa_R0040 in pSB1C3 from 2015</li>
−
<p> PCR of BBa_P0440 and BBa_R0040 in pSB1A2 from 2016 showed the right bands.
+
<li> digestion of BBa_P0440 and BBa_R0040 in pSB1C3 from 2015 showed negative results.</li>
−
<p> PCR amplification of Zinkfinger and Promotors parts. Digestion and ligation of the corresponding PCR-products.
+
<li> PCR of BBa_P0440 and BBa_R0040 in pSB1A2 from 2016 showed the right bands.</li>
−
</p>
+
<li> PCR amplification of Zinkfinger and Promotors parts. Digestion and ligation of the corresponding PCR-products.</li>
 +
</ul>
 
</div>
 
</div>
  
Line 82: Line 90:
 
<h1>October 2016</h1>
 
<h1>October 2016</h1>
 
<p><b>October 03 - October 09</b></p>
 
<p><b>October 03 - October 09</b></p>
−
<p>Determined that several parts were incorrectly labeled, which prevented correct assembly.
+
<ul>
−
<p>Inoculation of new L. johnsonii Culture
+
<li>Determined that several parts were incorrectly labeled, which prevented correct assembly.</li>
−
<p>Genomic DNA extraction of L.johnsonii, PCR on genomic DNA for homology arms (not successful)
+
<li>Inoculation of new L. johnsonii Culture</li>
−
<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).
+
<li>Genomic DNA extraction of L.johnsonii, PCR on genomic DNA for homology arms (not successful)</li>
−
<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.
+
<li>PCRs on gBlocks of Fru1, Fru2 with extension primers, adding BbsI-site (successful). Restriction and ligation of fragments. PCR on ligation reaction (not successful).</li>
−
<p> Amplification of Zinc Finger Parts via  PCR showed no the ligation of the corresponding parts.
+
<li> ligation of the PCR product of BBa_P0440 and BBa_R0040 were further amplified via PCR but did not show the right sized band.></li>
−
<p>PCR on putative Ketohexokinase, restriction, ligation into pSB1C3
+
<li> Amplification of Zinc Finger Parts via  PCR showed no the ligation of the corresponding parts.</li>
−
</p>
+
<li>PCR on putative Ketohexokinase, restriction, ligation into pSB1C3</li>
 +
</ul>
  
 
<p><b>October 10 - October 16</b></p>
 
<p><b>October 10 - October 16</b></p>
−
Fruitforce:
+
<ul>
−
<p>Restriction of ldh+pSB1C3 (successful)
+
<li>Restriction of ldh+pSB1C3 (successful)</li>
−
<p>Restriction of putative eSpCas9-BioBrick (not successful).
+
<li>Restriction of putative eSpCas9-BioBrick (not successful).</li>
−
<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).
+
<li>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).</li>
−
<P>PCR on Phosphofructokinase, restriction and ligation into pSB1C3, transformation (successful)
+
<li>PCR on Phosphofructokinase, restriction and ligation into pSB1C3, transformation (successful)</li>
−
<p>PCR on Phosphofructokinase, restriction and ligation with ldh+pSB1C3, transformation (successful)
+
<li>PCR on Phosphofructokinase, restriction and ligation with ldh+pSB1C3, transformation (successful)</li>
−
<p>Restriction of putative Sacc, ligation with ldh+pSB1C3 and transformation  (not successful)
+
<li>Restriction of putative Sacc, ligation with ldh+pSB1C3 and transformation  (not successful)</li>
−
<p>Restriction of Fru (successful), LDH-Promoter in pSB1C3 (successful).
+
<li>Restriction of Fru (successful), LDH-Promoter in pSB1C3 (successful).</li>
−
<p>Ligation of Fru and LDH-Promoter in pSB1C3 (not successful)
+
<li>Ligation of Fru and LDH-Promoter in pSB1C3 (not successful)</li>
−
<p>Restriction of LDH-Promoter Phosphofructokinase (successful), Ketohexokinase (successful), pTUM104 (successful), pNZ124+Term.(successful).
+
<li>Restriction of LDH-Promoter Phosphofructokinase (successful), Ketohexokinase (successful), pTUM104 (successful), pNZ124+Term.(successful).</li>
−
<p>Ligation and transformation of LDH-Promoter Phosphofructokinase with pNZ124+Term (not successful), Ketohexokinase with pTUM104 (not successful).
+
<li>Ligation and transformation of LDH-Promoter Phosphofructokinase with pNZ124+Term (not successful), Ketohexokinase with pTUM104 (not successful).</li>
−
</p>
+
</ul>
  
 
<b><p>October 17 - October 19</b></p>
 
<b><p>October 17 - October 19</b></p>
−
<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>
+
<ul>
 +
<li>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).</li>
 +
</li>
 
</div>
 
</div>
  
 
</div>
 
</div>
 
</html>
 
</html>

Latest revision as of 02:46, 20 October 2016

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).