2. Example construct we would want to construct in B.subtilis

3. Gel electro + purification Transformation
BGM Vector
Biobrick Assembly
DNA Synthesis
Transformation
PCR
PCR
PCR
PCR x4
Miniprep
Cloning
Cloning
Cloning
Cloning
Restriction Digest
Transformation
Ligation
Gel electro + purification
Transformation
Transformation
Transformation

4. Advantages
-Time - as assembly is parallel for many of the processes, and potentially cost is lower (not sure if PCR primers would make it more expensive though..)
-Can allow overwriting of sections of a device,
-Helps avoid the problems associated with cloning of small parts like RBS, as you can simply select for the antibiotic associated with addition of a part.
-Idea of software is also interesting, I wouldn’t know how to write the code but I think developing the rules of the program would be okay – i.e. have x amount of homology, recognising prefix + suffix, including scars into primers.

5. Issues to resolve:
Length required for homologous cross over. In this paper rely upon regions of 2kb, can we use smaller regions of 200bp?
There is no need for the biobrick standard anymore?
How can we standardise this:
Method of assembly (protocols),
Vector can be standardised,
No need for biobrick standard but parts can still be used in this method.
We can clone the devices we make out of the genome, but we would need to ensure that the assembly results in the biobrick scars for submission into the registry. (not a problem I don’t think as we can design our primers to include them)