Team:UPO-Sevilla/Notebook/07 13


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July, 13th

Production Team

Paola Gallardo. New parts fecA*, fecI, fecR*, PfecA, gltB and gltD*, their composite parts (fecI-fecR*, fecR*-PfecA y fecI-fecR*-PfecPG) and the vectors, that will be used in the ligation reactions later, were purified by GFX protocol. All of them were previously amplified. The first ones were obtained by PCR reaction from E. coli K12, and the others were extracted from the biobricks distribution 2010. After that, these parts were bound using restriction sites. Finally, they were transformed in E. coli and spread in LB+Cm plates.

David Caballero. Repetition of PCR reactions (in former experiments not completely satisfactory) to create biobricks: fecA*, fecR*, fecI*, PfecA, gltB, gltD**. PCR reactions were made in duplicate for each part. There were 19 PCR reactions in total: two for each simple part, one control without primers and two for the composite parts fecI-fecR*, fecR*-PfecA y fecI-fecR*-PfecA, taking advantage of the fact that these genes are consecutive in the E. coli genome.

PCR reactions were analyzed by 0.8% agarose gel electrophoresis. Results were not satisfactory: the gel overheated and ran for too long (1h and 30’), and fragments below 2 kbp ran off the gel. It is necessary to repeat the electrophoresis.

Assembly Team

First Fragments Assambly. We have named them “UPO+(a number)”. Consequently (So), in that way it would be easier to indentify the parts.

  • UPO 16 + UPO 3 (BBa_C0083 + BBa_B0015)
  • UPO 1* + UPO 19 (BBa_J23100 + BBa_J45319)
  • UPO 2* + UPO 13 (BBa_B0030 + BBa_E0040)
  • UPO 1* + UPO 2 (BBa_J23100 + BBa_B0030)
*Those fragments are oligonucleotids, that is the reason why they don’t need to be digested
  1. Digestion of the fragments we require
  2. Purification of the DNA with the kit GFX ( DNA Purification thanks to Kit GFX. DNA Purification by using the KIT GFX. DNA Purification using the Kit GFX.)
  3. Time to ligate the DNA. They all use the lineal vector pSB1C3, except for UPO1+UPO2 (UPO2 it is an ampiciline resistant vector)
  4. Transformation in DH5α
  5. Cultures are made on plates with Cm and Am
  6. While waiting, (we prepare the)preparation of LB medium and agar to make electrophoresis

We cannot use the LacZ biobrick, due probably to the fact that this fragment is defective. Under those circumstances Its transformation failed

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