Difference between revisions of "Team:Uppsala/Notebook"

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<ul><li>
 
<ul><li>
 
Made an attempt to extract the Nah7 plasmid using the designed primers, including gel electrophoresis of the PCR product. The gel showed that the PCR had not worked.</li></ul>
 
Made an attempt to extract the Nah7 plasmid using the designed primers, including gel electrophoresis of the PCR product. The gel showed that the PCR had not worked.</li></ul>
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<ul><li>
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PICTURE 2!!!
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</li></ul>
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<ul><li>
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Since the NoKoGen biobricks had already been improved, transformation were done with the improved biobricks of rhlA, rhlB, and with RBS and the BBa_J23101 promoter from the kit into DH5α. After that plasmid preparation were done. This as a preparation for building the biosurfactant construct.</li></ul>
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</p>
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<h4>Week 4: (29-5 June/July)</h4>
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  <p><ul><li>
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A first attempt to remove the T7-promoter from the biobricks with CueO, CotA and catechol 1,2-dioxygenase was done. The products were run on a gel and this showed that the PCR was unsuccessful.</li></ul>
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<ul><li>
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Picture 3. of gel!!!
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</li></ul>
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<ul><li>
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A transformation on the ligations with CueO, CotA and catechol 1,2 dioxygenase with the HlyA-tag gene were transformed but with no results. The assemblies were redone, but a colony PCR indicated that the assembly was unsuccessful.
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</li></ul>
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<ul><li>
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Assembled NahR with the reporter genes received from Erik Gullberg.
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</li></ul>
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<ul><li>
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Assembling rhlA, rhlB, with RBS and the BBa_J23101 promoter.
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</li></ul>
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<ul><li>
 +
Assembled plasmids with chromoproteins to plasmid backbones and then transformed them into DH5α. Made bactoart using the transformed bacteria.</li></ul>
 
</p>
 
</p>
  

Revision as of 00:16, 19 September 2015


Notebook

Week 1: (8-14 June)

  • Primers were designed for PCR extraction of the naphthalene upper pathway of the Nah7 plasmid from Pseudomonas putida, and to remove T7-promoters from CueO, CotA and catechol 1,2-dioxygenase biobricks. We also designed primers to do overlap extension PCR to remove the scar that was created when attaching the HlyA-tag with the CueO, CotA and catechol 1,2-dioxygenase genes.
  • Received the JapLac sequence from professor Kataoka.
  • Stock solutions and agar plates with and without antibiotic resistance were made.

Week 2: (15-21 June)

  • Primers were designed for site directed mutagenesis to eliminate the restrictions sites for improving the NoKoGen biobricks, and primers were also designed for restriction free cloning to assemble the HlyA-tag with the CueO, CotA and dioxygenase genes, without creating a scar.
  • rhlA and rhlB genes from the DNA distribution kit were transformed into E.coli DH5α and plasmid preparations were made.

Week 3: (22-28 June)

  • Transformations were done to insert biobricks with the enzymes CueO, CotA, catechol 1,2-dioxygenase and the HlyA-tag genes into DH5α. Frozen stock and plasmid preparations were made of these.
  • Assembled the HlyA-tag with CueO, CotA and dioxygenase with 3A assembly.
  • Transformed the NahR construct, dTomato and super yellow fluorescent protein 2 (SYFP2), as well as plasmid prepared the transformed DNA and evaluated with PCR followed by further evaluation by agarose gel electrophoresis.
  • Designed primers for sequencing of the Nah7 pathway, as well as primers flanking the entire pathway.
  • Made an attempt to extract the Nah7 plasmid using the designed primers, including gel electrophoresis of the PCR product. The gel showed that the PCR had not worked.
  • PICTURE 2!!!
  • Since the NoKoGen biobricks had already been improved, transformation were done with the improved biobricks of rhlA, rhlB, and with RBS and the BBa_J23101 promoter from the kit into DH5α. After that plasmid preparation were done. This as a preparation for building the biosurfactant construct.

Week 4: (29-5 June/July)

  • A first attempt to remove the T7-promoter from the biobricks with CueO, CotA and catechol 1,2-dioxygenase was done. The products were run on a gel and this showed that the PCR was unsuccessful.
  • Picture 3. of gel!!!
  • A transformation on the ligations with CueO, CotA and catechol 1,2 dioxygenase with the HlyA-tag gene were transformed but with no results. The assemblies were redone, but a colony PCR indicated that the assembly was unsuccessful.
  • Assembled NahR with the reporter genes received from Erik Gullberg.
  • Assembling rhlA, rhlB, with RBS and the BBa_J23101 promoter.
  • Assembled plasmids with chromoproteins to plasmid backbones and then transformed them into DH5α. Made bactoart using the transformed bacteria.