"

From 2010.igem.org

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:

• Whilst waiting for DNA from the parts registry we tried to obtain large amounts of the constitutive GFP biobrick
• 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.
• Thus we tried transforming two further strains; XL1 Blues and commercial NovaBlues (see table for details)

XL1-blue positive control	100μL cells + 2μL known plasmid solution
XL1-blue negative control	100μL cells (untransformed)
XL1-blue + 2009 biobrick	100μL cells + 5μL 2009 biobrick
XL1-blue + 2010 biobrick	100μL cells + 5μL 2010 biobrick
Nova blue + 2009 biobrick	100μL cells + 5μL 2009 biobrick
Nova blue + 2010 biobrick	100μL cells + 5μL 2010 biobrick

• Only the commercial cells were successful and provided us with colonies
• Colonies 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 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.
• Once the DNA had arrived, the same procedure was followed to obtain large amounts of BBa_K216005.