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From 2010.igem.org

Constructing Our Biobrick - K381001

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
• Phosphatase treatment of the vector and gel extraction of the digests
• 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, typically transforming 50μL of cells with 2μL of DNA
• 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_E0840 and BBa_E0240 were digested with Xba1 and Pst1 using the following mix:
  • 30μL DNA
  • 0.5μL BSA 100x
  • 5μL NEB Buffer 3
  • 1μL Pst1
  • 1.3μL Xba1
  • 12.2μL ddH2O

• BBa_K216005 was digested with Spe1 and Pst1 using the following mix:
  • 30μL DNA
  • 0.5μL BSA 100x
  • 5μL NEB Buffer 2
  • 1.3μL Pst1
  • 1μL Spe1
  • 12.2μL ddH2O

• Each digest was left for 2 hours at 37°C