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Team:BCCS-Bristol/Calendar-Notebook/13 August 2008
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==BioBrick Transformation== | ==BioBrick Transformation== | ||
The self-made electro competent ''E. coli'' DH5α cells were tested twice. The transformation efficiency with pUC19 was 4.45 x 10<sup>8</sup> cfu/µg and 5.67 x 10<sup>8</sup> cfu/µg. The first attempt with BioBrick DNA failed, but this might be due to a too low cell amount compared to all the other decanted tubes. In the following transformations, the incubation of the punched paper disc with the DNA was varied in time (3 h and 5.5 h). It seems that a longer incubation increases the transformation efficiency. This has to be confirmed, since the transformations were conducted by different persons. Thereby, next to the [http://partsregistry.org/Part:BBa_J63005 yeast ADH1 promoter] another BioBrick ([http://partsregistry.org/Part:BBa_J63001 enhanced version of EYFP, yeast-optimized YFP]) was tested because the VF2-VR value of [http://partsregistry.org/Part:BBa_J63005 yeast ADH1 promoter] we got didn't coincide with the value given on the iGEM page. | The self-made electro competent ''E. coli'' DH5α cells were tested twice. The transformation efficiency with pUC19 was 4.45 x 10<sup>8</sup> cfu/µg and 5.67 x 10<sup>8</sup> cfu/µg. The first attempt with BioBrick DNA failed, but this might be due to a too low cell amount compared to all the other decanted tubes. In the following transformations, the incubation of the punched paper disc with the DNA was varied in time (3 h and 5.5 h). It seems that a longer incubation increases the transformation efficiency. This has to be confirmed, since the transformations were conducted by different persons. Thereby, next to the [http://partsregistry.org/Part:BBa_J63005 yeast ADH1 promoter] another BioBrick ([http://partsregistry.org/Part:BBa_J63001 enhanced version of EYFP, yeast-optimized YFP]) was tested because the VF2-VR value of [http://partsregistry.org/Part:BBa_J63005 yeast ADH1 promoter] we got didn't coincide with the value given on the iGEM page. | ||
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| + | ==Agar Plug Assay== | ||
| + | Evaporation and currents that were occurring in previous attempts at the agarose-in-plug assay were eliminated by ensuring water potentials of plug and chemotaxis buffer were as similar as possible. | ||
| + | Both the beads and the bacteria were found to sink to the bottom of the chamber shortly after inoculation. This makes the model much simpler, as everything can be modelled in 2D. | ||
| + | We showed that beads need to be left in bacteria for around 30mins in order for significant bead movement to occur. We currently have not determined if this is because the bacteria form a layer under the beads which stops them sticking to the slide, or if adherence itself is needed for bead movement. | ||
| + | We determined the diffusion speed of bromophenol blue dye (which is three times larger than aspartate), and concluded that by 30mins aspartate would have diffused throughout the slide. | ||
Revision as of 10:41, 19 August 2008
BioBrick Transformation
The self-made electro competent E. coli DH5α cells were tested twice. The transformation efficiency with pUC19 was 4.45 x 108 cfu/µg and 5.67 x 108 cfu/µg. The first attempt with BioBrick DNA failed, but this might be due to a too low cell amount compared to all the other decanted tubes. In the following transformations, the incubation of the punched paper disc with the DNA was varied in time (3 h and 5.5 h). It seems that a longer incubation increases the transformation efficiency. This has to be confirmed, since the transformations were conducted by different persons. Thereby, next to the [http://partsregistry.org/Part:BBa_J63005 yeast ADH1 promoter] another BioBrick ([http://partsregistry.org/Part:BBa_J63001 enhanced version of EYFP, yeast-optimized YFP]) was tested because the VF2-VR value of [http://partsregistry.org/Part:BBa_J63005 yeast ADH1 promoter] we got didn't coincide with the value given on the iGEM page.
Agar Plug Assay
Evaporation and currents that were occurring in previous attempts at the agarose-in-plug assay were eliminated by ensuring water potentials of plug and chemotaxis buffer were as similar as possible. Both the beads and the bacteria were found to sink to the bottom of the chamber shortly after inoculation. This makes the model much simpler, as everything can be modelled in 2D. We showed that beads need to be left in bacteria for around 30mins in order for significant bead movement to occur. We currently have not determined if this is because the bacteria form a layer under the beads which stops them sticking to the slide, or if adherence itself is needed for bead movement. We determined the diffusion speed of bromophenol blue dye (which is three times larger than aspartate), and concluded that by 30mins aspartate would have diffused throughout the slide.
