Team:BCCS-Bristol/Wetlab/K381001 Construction

From 2010.igem.org

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Generating large quantities of DNA:
Generating large quantities of DNA:
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* Whilst waiting for DNA from the parts registry we tried to obtain large amounts of the constitutive GFP biobrick
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* The first stage was to transform commercial NovaBlue competent cells with our component biobricks
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* We initially tried transforming MG1655s with DNA from the kit plate but were unsuccessful. Suspected the problem was down to cells not being competent enough and insufficient DNA in kit plate.
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* Colonies from these transformations were then selected and used to grow overnights - using a wire loop to inoculate 5mL of LB medium (5μL of Ampicillin also added)
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* Thus we tried transforming two further strains; XL1 Blues and commercial NovaBlues (see table for details)
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* Combined 3mL of BBa_K216005, BBa_E0840 and BBa_E0240 cell cultures in 1.5mL eppendorf tubes twice - total of 6mL combined cell culture for each biobrick. Performed a miniprep on the combined cultures using a Qiagen "QIAprep Spin Miniprep Kit". Eluted 100μL of solution containing BBa_K216005, BBa_E0840 and BBa_E0240 biobricks in high concentration.
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{|style="border-collapse: separate; border-spacing: 0; border-width: 1px; border-style: solid; border-color: #000"
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Digesting Component Biobricks:
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!style="border-style: solid; border-width: 1px"| XL1-blue positive control
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* BBa
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!style="border-style: solid; border-width: 1px"| 100μL cells + 2μL known plasmid solution
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!style="border-style: solid; border-width: 1px"| XL1-blue negative control
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!style="border-style: solid; border-width: 1px"| 100μL cells (untransformed)
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!style="border-style: solid; border-width: 1px"| XL1-blue + 2009 biobrick
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!style="border-style: solid; border-width: 1px"| 100μL cells + 5μL 2009 biobrick
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!style="border-style: solid; border-width: 1px"| XL1-blue + 2010 biobrick
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!style="border-style: solid; border-width: 1px"| 100μL cells + 5μL 2010 biobrick
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!style="border-style: solid; border-width: 1px"| Nova blue + 2009 biobrick
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!style="border-style: solid; border-width: 1px"| 100μL cells + 5μL 2009 biobrick
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!style="border-style: solid; border-width: 1px"| Nova blue + 2010 biobrick
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!style="border-style: solid; border-width: 1px"| 100μL cells + 5μL 2010 biobrick
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|}
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* Only the commercial cells were successful and provided us with colonies
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* Colonies then selected and used to grow overnights - using a wire loop to inoculate 5mL of LB medium (5μL of Ampicillin also added)
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* Combined 3mL of cell culture from these overnights in 1.5mL eppendorf tube 4 times - total of 12mL combined cell culture. Performed a miniprep on the combined culture using a Qiagen "QIAprep Spin Miniprep Kit". Eluted 200μL of solution containing [http://partsregistry.org/Part:BBa_I13522 BBa_I13522] biobrick in high concentration.
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* Once the DNA had arrived, the same procedure was followed to obtain large amounts of BBa_K216005.
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Revision as of 11:30, 17 September 2010

Having decided on our biobrick design, our first task was to aquire the necessary parts and construct our first biobrick.

BBa_I13522 was taken from well 8A kitplate 2 of the 2010 distribution BBa_K216005 was requested from the parts registry

Construction of the biobrick can be broadly broken down into

  • Generating larger quantities of both our component biobricks
  • Digesting each of these biobricks
  • Ligating together the desired parts to create a new biobrick
  • Generating larger quantities of this biobrick, checking and sequencing


Generating large quantities of DNA:

  • The first stage was to transform commercial NovaBlue competent cells with our component biobricks
  • Colonies from these transformations were then selected and used to grow overnights - using a wire loop to inoculate 5mL of LB medium (5μL of Ampicillin also added)
  • Combined 3mL of BBa_K216005, BBa_E0840 and BBa_E0240 cell cultures in 1.5mL eppendorf tubes twice - total of 6mL combined cell culture for each biobrick. Performed a miniprep on the combined cultures using a Qiagen "QIAprep Spin Miniprep Kit". Eluted 100μL of solution containing BBa_K216005, BBa_E0840 and BBa_E0240 biobricks in high concentration.

Digesting Component Biobricks:

  • BBa
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